Version 5.
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Table of Contents 1. Introduction ............................................................. 1 Overview of Microplate Manager..........................................................1 Types of Files ......................................................................................3 New Features in Version 5.2 ................................................................4 2. Installation and Setup ............................................. 5 Instrument Specifications ..........................
Setting Filters .................................................................................... 11 Ultramark and Ultramark EX Setup.........................................................12 Model 550 Setup .....................................................................................12 Preferences....................................................................................... 13 Sample Data Files.............................................................................
Plate Loading in a Multiple Plate Protocol............................................... 44 Saving Files from Multiple Plate Readings.............................................. 46 Absorbance Delay and Ratio Delay ........................................................ 49 5. Templates .............................................................. 53 Opening a Template ..........................................................................54 Opening an Existing Template...............................
Kinetic Correlation Coefficient Report ...................................................102 Kinetic Standard Errors Report..............................................................102 Kinetic Plots Report ...............................................................................103 Kinetic Zoom Plot ..................................................................................104 Report Labels..................................................................................
Appendix A. Menus .................................................. 125 Appendix B. Glossary .............................................. 131 Appendix C. Ordering Information......................... 133 Appendix D. Warranty.............................................. 135 Index ..........................................................................
Chapter 1. Introduction 1. Introduction This manual assumes that you are familiar with your computer and standard commands and functions associated with the Windows® operating system, such as opening, closing, and saving files, and moving and clicking your mouse. Some of the features and functions in Microplate Manager will be slightly different depending on which microplate reader you are using (e.g., Benchmark Plus, Ultramark, Model 550, etc.). These differences are noted in the text.
Microplate Manager User Guide Select Type of Reader Select Type of Reading (Endpoint, Kinetic, or Multiple Plate Protocol) Define or Select Microplate Template Take Reading Generate and Format Reports Print/Export Data Figure 1. Workflow of Microplate Manager Microplate Manager can perform three general types of microplate readings. These are called “Protocols.” • Endpoint Protocols are used to acquire a single absorbance reading from each well.
Chapter 1. Introduction template for your microplate. Finally you take the reading. If you want to use the particular Protocol you’ve created again, you can save it as a file. After the absorbances have been read, you can save them as a Data File and display them in a selection of reports. Your data can be viewed, printed, or exported to other applications. Types of Files There are two types of files used by Microplate Manager. Protocol Files contain the settings for reading a microplate.
Microplate Manager User Guide Data Files Data Files contain the data from a microplate reading. They have the extension .mpm. For an Endpoint reading, this data consists of the individual absorbance readings from the wells on the microplate. For a Kinetic reading, this data is the time course series of the individual absorbance readings. A Multiple Plate reading creates a new Data File for each plate; each file contains Endpoint absorbance measurements.
2. Installation and Setup Instrument Specifications Computer Microplate Manager software is compatible with Windows® 95, 98, ME, NT, 2000, and XP. The minimum computer requirements for running Microplate Manager are: Processor: Pentium 166 Memory: 16 MB RAM Disk size: 200 MB Monitor: 1024 x 768 resolution, 256 colors. Microplate Readers Microplate Manager 5.2 supports Bio-Rad’s Benchmark Plus, Benchmark, Ultramark, Ultramark EX, Model 550, and Model 680 microplate readers.
Microplate Manager User Guide Connecting the Microplate Readers to the Host Computer A serial cable is required to connect the Benchmark Plus, Benchmark, Model 550, and Model 680 microplate reader with the computer. Make sure the computer and all devices are turned off before making or changing cable connections. See your specific hardware manual for details. A SCSI cable and card are required to connect the Ultramark and Ultramark EX to the computer.
Chapter 2. Installation and Setup C:\Program Files\Bio-Rad\Microplate Manager. You can select a different directory when prompted to do so by the installer. The installer will place a startup icon on your desktop and create a Microplate Manager directory on your Windows® Start menu. When the installer is finished, click on the Finish button.
3. Getting Started Menus, Toolbars, and Other Features Menu Bar Microplate Manager has a standard menu bar with pulldown menus that include all the commands and functions in the software. The available menus and menu items are different depending on the specific actions you are performing. For example, when the software first opens, the menus will include basic file opening and set-up features. When you are formatting a template, the menus will change to include functions for formatting a template.
Microplate Manager User Guide Right-click Commands You can access many of the commands associated with the action you are currently performing by using the right mouse button. For example, if you are in the process of defining an assay on your template, position your cursor over a well in the template and click the right mouse button to display many of the commands associated with that action. The commands are displayed in a context menu next to the cursor. Click right mouse button Figure 4.
Chapter 3. Getting Started Selecting a Reader To select an instrument, go to the FILE menu and select Select Reader. Figure 5. Select Reader dialog box. From the pull-down menu, select the appropriate reader and click on OK. If you select a reader that is not connected or is otherwise unavailable, you will receive a warning message when you attempt to open a Protocol window. Microplate Manager does not attempt to communicate to the reader until you open a Protocol window.
Microplate Manager User Guide Ultramark and Ultramark EX Setup With a Protocol window open, select Instrument Setup from the FILE menu. Figure 6. Ultramark Setup dialog box. For each filter, enter the appropriate wavelength. The wavelengths of the filters that are shipped with the Ultramark are included with your Ultramark documentation. If you change any of the filters, you will need to change these settings. Click on OK to implement your changes.
Chapter 3. Getting Started Figure 7. Model 550 Setup dialog box. For each filter, enter the appropriate wavelength in the dialog. The wavelengths of the filters that are shipped with the Model 550 are included with the reader documentation. If you change any of the filters, you will need to change these settings. Click on OK to implement your changes. Preferences To set the user-defined preferences for Microplate Manager, select Preferences from the FILE menu. Figure 8. Preferences dialog box.
Microplate Manager User Guide In the Preferences dialog, enter or select the Default File Location for opening and saving files, and enter or select the Custom Report Template File Location for opening and saving custom report templates. To select the directory, click on the “...” button next to the text field and scroll through the directory tree. You can also type the full directory path and name directly in the field.
Chapter 3. Getting Started Figure 9. Examples directory. Clicking on any of these Data Files will open Microplate Manager and display the template for that file. Then you can view the sample data using the commands described in Chapter 6. Opening and Saving Files Microplate Manager has two basic file types: Protocol Files and Data Files. Protocol Files have a .kpr, .epr., or .mpr extension. Data Files have an .mpm extension. To open an existing Protocol File or Data File, select Open...
Microplate Manager User Guide Importing Data This version of Microplate Manager will read files from the previous version (5.0.x). Tab-delimited and comma-separated data are also supported (TXT, CSV, LOT, ASC). From Kinetic Collector PC, Data Files in matrix format, row format, or column format may be imported (MTX, ROW, or COL).
Chapter 3. Getting Started Scenario 1. Endpoint Assay with Previously Formatted Template You want to perform an Endpoint assay using a template layout that you have already defined. From the FILE menu, you select New Endpoint Protocol. This opens the Endpoint Protocol dialog box. You enter Mix Time, Initial Wait, and Incubator settings for the reader. Figure 10. Performing an Endpoint reading.
Microplate Manager User Guide Concentration Report from the VIEW menu). You select Print... from the FILE menu to print this report. You decide that you will be repeating this operation frequently, so you go back to the Endpoint Protocol dialog box. The Reports... button opens a dialog box in which you specify the reports to view and/or print after each reading. Select the Unknown Concentration Report for printing, and click on OK.
Chapter 3. Getting Started After inserting your plate into the reader, you click on Run and the reader begins collecting data. At this point, both the template and the Kinetic Plots Report automatically pop up. The Kinetic Plots Report shows a graph of absorbance versus time for each well. As each reading is completed, the graphs are updated. After all of the data has been read, you can display any relevant report (Limit, Control, etc.) by selecting it from the VIEW menu.
Microplate Manager User Guide Figure 12. Comparing external and internal standards.
4. Protocols Protocols contain the settings for controlling the microplate reader. To read a microplate, you must first open a Protocol window and specify the settings. Some of the features and functions in the Protocols are different depending on the type of microplate reader you are using. These differences are noted in the text. The figures depict some functions that are only available with certain microplate readers.
Microplate Manager User Guide If no reader is found, you will receive a warning message. Make sure the reader is turned on, and check all the connections to the reader. See the microplate reader hardware manual for more details. Creating/Opening a Protocol Creating a New Protocol To create a new Protocol, select New Kinetic Protocol, New Endpoint Protocol, or New Multiple Plate Protocol from the FILE menu. This will open the dialog box for that particular type of Protocol.
Chapter 4. Protocols Figure 13. Example of a Protocol dialog box (Ultramark version shown). Each Protocol dialog box has numerous settings for controlling the microplate reader. Some settings are the same for all Protocols, while others are unique to the specific type of Protocol. Also, some settings are only available with certain types of microplate readers; these reader-specific settings are noted in the descriptions that follow.
Microplate Manager User Guide Selecting a Template Each Protocol File includes a microplate template that specifies the number of wells to be read, as well as any additional formatting information you want to include (locations of standards, unknowns, controls, etc.). This template is copied into the Data File when a reading is taken. From within the Protocol dialog box, you can either create a new template or pick an existing template.
Chapter 4. Protocols Figure 14. Selecting a plate type (Benchmark Plus types shown). The available plate types are different for the Benchmark Plus and Ultramark. Custom Plate Types (Ultramark Only) Ultramark users can click on the Custom Plate Type... button to create a new plate type. Custom plate types are listed in parentheses. Figure 15. Selecting a custom plate type (Ultramark only). Clicking on Custom Plate Type... is the same as selecting New Template > Custom... from the FILE menu.
Microplate Manager User Guide or Save Protocol As... from the FILE menu, or click on the Save button on the main toolbar. Protocols are not automatically saved when you click on Run. To preserve changes to a Protocol File, you must save it. When a template is saved in a Protocol File, it will remain there until you select a different template. If you create or edit a template in one Protocol and want to copy it to another Protocol, you must first save it as a separate Data File.
Chapter 4. Protocols Figure 17. Measurement wavelength settings in the Protocol dialog box (Benchmark Plus version shown). For the Benchmark Plus, you can adjust the monochrometer by clicking on the updown arrows next to each filter field, or you can enter values from 340 to 800 nm directly in the fields. For microplate readers other than the Benchmark Plus, the available filters are listed in the pulldown menus. (See page 11 for instructions on how to set the filter wavelengths for your microplate reader.
Microplate Manager User Guide Incubator Controls (Not Available for Model 550) To use the incubator in the microplate reader, click on the Incubator On checkbox in the Protocol dialog box. Figure 18. Incubator settings. The incubator will warm up to the Setpoint temperature and maintain it throughout the reading. For all readers, the default setpoint is 37.0°C. Otherwise: • For the Benchmark, the 37.0°C setpoint is fixed. • For the Model 680 and Benchmark Plus, the setpoint range is 25.0–45.0°C.
Chapter 4. Protocols In Multiple Plate Protocols, the initial wait time only applies to the first plate in the batch to be read. Mix Settings Mix Time specifies the length of time that a plate is shaken before the reading is taken. Enter a mix time in seconds. The maximum mix time ranges from 99 seconds to 999 seconds, depending on the type of reader. When you click on Run, the reader will shake the plate for the specified mix time before taking a reading.
Microplate Manager User Guide Figure 20. Scan Well settings (Benchmark Plus only). In the Scan Well dialog box, specify the Row and Column of the well to be scanned. Next, specify the wavelength range by entering values in the Shortest Wavelength (min = 340 nm) and Longest Wavelength (max = 800 nm) fields. The Step Size is the increment between scanning wavelengths, in nanometers. Finally, place the plate to be scanned in the reader and click on the Scan Well button.
Chapter 4. Protocols Figure 21. Well Scan Graph (Benchmark Plus only). You can aim your cursor at any point in the graph and display the wavelength and OD values of that point in the fields in the lower right of the window. Click on Copy Graph View to place a bitmap image of the graph on the computer clipboard. Click on Copy Graph Data to copy the data points along the graph to the computer clipboard. Print will send a copy of the graph image to the specified printer.
Microplate Manager User Guide Figure 22. Reports dialog box for Endpoint and Multiple Plate Protocols. At least one non-custom report must be selected to display after a reading. For Endpoint and Multiple Plate Protocols, the Raw Data Report is the default report to be displayed. For Kinetic Protocols, the Kinetic Plots Report is the default report. You can select one or more reports using the checkboxes.
Chapter 4. Protocols For example, if the labels “Date” and “Operator” are created in a Protocol File, then when that Protocol is run, a dialog box will pop up after plate reading prompting you to type the date and the operator’s name. Both the label (e.g., Date) and the label contents (e.g., July 4, 1999) are printed on the report. In the Reports dialog box, click on Labels... to open the Define Labels dialog box. Figure 23. Define Labels dialog box. See page 105 for information about defining labels.
Microplate Manager User Guide If you don't fill in the labels, you will be prompted to do so when you run the Protocol. You can also define and fill out labels after the Protocol has been run, when the data is displayed. Labels created after a Protocol has been run cannot be saved with the Protocol. Running a Protocol After all the parameters have been set, click on the Run button to begin taking a reading.
Chapter 4. Protocols Protocols are not automatically saved when you click on Run. To preserve changes to a Protocol File, you must save it. To save a Protocol, select Save Protocol or Save Protocol As... from the FILE menu or click on the Save button on the main toolbar. Enter a name for the Protocol in the dialog box. The default extensions for Kinetic, Endpoint, and Multiple Plate Protocol Files are .kpr, .epr, and .mpr, respectively.
Microplate Manager User Guide Absorbance Delay (Ratio Delay) button Initial wait field Figure 26. Features of an Endpoint Protocol dialog box. Absorbance Delay and Ratio Delay Absorbance Delay is available in Endpoint and Multiple Plate Protocol windows. Ratio Delay is a form of Absorbance Delay that becomes available if you have elected to ratio a dual wavelength reading (see page 27). Absorbance Delay delays data collection until a well that you select reaches a specified level of absorbance.
Chapter 4. Protocols Figure 27. Absorbance Delay dialog box. In the Absorbance Delay dialog box, click on the Use Absorbance Delay checkbox to enable this feature. Select the row and column number of the well whose absorbance you want to monitor from the pull-down lists. Under Acquire when the reading is, specify an absorbance that is above or below a certain OD value.
Microplate Manager User Guide Figure 28. Ratio Delay dialog box. Kinetic Protocol Features Kinetic Protocols are used to acquire a series of absorbance readings from each well over a time period that you specify. You can use these Protocols to calculate reaction velocities. To run a new Kinetic Protocol, select New Kinetic Protocol from the FILE menu.
Chapter 4. Protocols Reading sets Analysis options Figure 29. Features of a Kinetic Protocol dialog box. The controls common to all Protocols are described on page 23. The controls specific to Kinetic Protocols are described below. Reading Sets A reading set consists of the number of times a plate is read, the delay between each reading, and (if desired) the mixing time before each reading. You can specify up to five reading sets per Protocol.
Microplate Manager User Guide Enter the number of readings you want to take in the Readings column. Next enter the interval between the start of two consecutive readings in the Interval column. Note that this is the minimum time necessary for one complete reading plus any mixing time you specify. For example, when using the Ultramark with a standard 12 × 8 (96-well) plate, the smallest interval you can specify is 7 seconds. For custom plate sizes, the smallest interval you can specify is 450 seconds (7.
Chapter 4. Protocols Figure 30. Analysis Options in the Kinetic Protocol dialog box. The settings in this dialog box will only affect the analysis of your data. They will not change the raw data that is gathered. Time and Absorbance Limits In this dialog, you can specify a time range and/or an absorbance range for your data points. If time limits and/or absorbance limits are set, then only data points within these limits will be used to calculate the reaction velocity.
Microplate Manager User Guide To set absorbance limits, select the appropriate checkbox(es) and enter ODs in the appropriate field(s). Note that if you have elected to ratio a dual wavelength measurement in the main Kinetic Protocol window (see page 26), the Analysis Options dialog box will contain fields to set the ratio limits of the reading (enter a measurement/reference ratio in each field). Figure 31. Ratio limits in the Analysis Options dialog box.
Chapter 4. Protocols For cases where the absorbances decrease with time, the velocities will be negative. In these cases, select the Negative Kinetics checkbox. If the velocities are negative, then the velocity or maximum velocity axis of the Standard Curve Report is constrained to linear unless the Negative Kinetics box is checked (logarithms cannot be taken of negative values).
Microplate Manager User Guide Absorbance Delay (Ratio Delay) button File format Plate loader Multiple Plate settings Figure 32. Features of a Multiple Plate Protocol dialog box. The controls common to all Protocols are described starting on page 23. The controls specific to Multiple Plate Protocols are described below.
Chapter 4. Protocols Without a Plate Loader For readers not connected to a plate loader, enter the number of plates in the Num of Plates field. Then, when you click on Run, you will be prompted to insert the next plate in the sequence after each plate has been read. With a Plate Loader (Benchmark Plus and Ultramark only) Benchmark Plus and Ultramark users can connect their readers to Bio-Rad’s Twister plate loader.
Microplate Manager User Guide From this dialog box you can enter the main Twister control panel by clicking on the Twister Configuration/Training button. See the Twister User Guide for instructions on configuring the Twister using this panel. After you exit the Twister control panel, you will have to click on the Run button again to reenter the Plate Handler Options dialog box.
Chapter 4. Protocols Figure 34. Save Format selections. MPM Format Only Under Save Format, select MPM format only. When you click on Run, you will be prompted to select a base file name for the resulting Data Files. File names are constructed by appending two digits to the base file name (e.g., test-01.mpm, test02.mpm, etc.). Enter the base file name and select the directory in which to save the files.
Microplate Manager User Guide Figure 35. Multiplate Export Options dialog box. This dialog gives you a number of options for exporting your plate data. Under File Selection, you can export a separate text file for each plate read (Per plate), or create a single text file for all the plates read in a multiplate run (Per run). You can also append the run data to another text file by selecting Master file, and clicking on the Select File button to select the master Data File to append to.
Chapter 4. Protocols Under Data Labeling, select if and how you want to label the values reported for the wells in a plate. You can export additional information for each plate by selecting from the list of Additional Labels. Double-click on the desired plate label in the Available column to move it to the Selected column. Double-click on an item in the Selected column to move it back to the Available column.
Microplate Manager User Guide Figure 36. Absorbance Delay dialog box. In the Absorbance Delay dialog box, click on the Use Absorbance Delay checkbox to enable this feature. Select the row and column number of the well whose absorbance you want to monitor from the pull-down lists. In the Acquire when the reading is section, specify an absorbance that is above or below a certain OD value.
Chapter 4. Protocols Figure 37. Ratio Delay dialog box.
5. Templates Templates allow you to specify the number and layout of the wells on your microplates. You use this layout information when analyzing your data and organizing it in your final report(s). The following information can be stored in a template: • Number and location of assays on the plate. • Well type (i.e., standard, unknown, positive or negative control, blank, or undefined). • Replicate groups (if any) for standards and unknowns within each assay.
Microplate Manager User Guide It is useful to format your template before reading the microplate so that this information can be included in the Protocol File. This allows reports to be automatically generated, displayed, and printed immediately after plate reading. Note that the template does not affect how a plate is read. All wells in the microplate will be read by the reader, regardless of any template formatting.
Chapter 5. Templates Benchmark Plus and Ultramark users can select from one of three standard plate types: 12 × 8, 24 × 16, or 48 × 32. Benchmark, Model 550, and Model 680 users can only select the standard 12 × 8 plate type. If you are using the Ultramark, you can also define your own plate type size by selecting Custom. Select Custom to select/add a different plate type Figure 38. Selecting a template.
Microplate Manager User Guide Figure 39. Creating a custom plate type. In this dialog box, if the template format you want is listed, simply double-click on it to select it (or click on it once to highlight it, then click on the Select button to select it). 56 • To copy a format, click on the format name, then click on Copy. The copy will have the original format name plus a “:1” extension. You can then modify the copy (see below).
Chapter 5. Templates Figure 40. Defining the format of a plate. In the Define Plate Format dialog box, you can enter the numbers of Columns and Rows in your microplate in the appropriate fields. You can also choose how your columns and rows should be labeled—with Letters or Numbers. The default labeling is numbers for columns and letters for rows; however, if the number of rows exceeds 26, numbers will be substituted for letters. You can enter a different name for your plate in the Plate Name field.
Microplate Manager User Guide Figure 41. Entering the physical dimensions of your plate. In the Physical Dimensions dialog box, you can enter the distance in millimeters from the edges of the plate to the centers of the corner wells. Entering the offset distances for the top left well (x1, y1) and bottom right well (x2, y2) will provide the dimensions for the entire plate. Refer to the diagram in the dialog box for guidance on which distance to enter into which field.
Chapter 5. Templates When you have specified the physical dimensions of your plate, click on the OK button. Template Features When you select a new template from the FILE menu (or from within one of the Protocol dialog boxes), a blank template will open, displaying the layout grid you selected and the template toolbar. Toolbar Grid Figure 42. Blank 12 x 8 template. Using the template toolbar, you define the wells that contain your standards, unknowns, blanks, and controls.
Microplate Manager User Guide Template Size You can display a template in three sizes. Under the VIEW menu, select Show Full Size, Show Compact, or Show Tiny. The smaller sizes are useful for viewing larger templates. If a template is too large to fit in the display window, it will be displayed with scroll bars along the right side and bottom of the template window.
Chapter 5. Templates Figure 44. Well Info dialog box. The info box shows the number, type, and reading of the well you selected. It also lists all the wells in its replicate group, and shows the dilution for the replicate group. You can enter a new dilution here, and the change will be reflected throughout the application. You can only display the properties for one well at a time. Formatting a Template After you have opened a template (see page 54), you are ready to format it.
Microplate Manager User Guide Figure 45. Format menu and template toolbar. Some additional assay-related commands are located on the EDIT and ASSAY menus. Undo and Redo Formatting Commands If you make a mistake while formatting, select Undo Formatting from the EDIT menu. This will undo the last formatting change you made to the plate template. Once undone, the formatting can be redone by choosing Redo Formatting from the EDIT menu.
Chapter 5. Templates subsequent assays, they appear as different colors. Independent statistical analysis and data reduction are performed for each assay. To define an assay, click on the Assay button on the template toolbar (or select Assay from the FORMAT menu). The cursor will change into the Assay tool. Then drag the mouse to select all wells in the assay. As you drag, a box will appear on the template marking the selected wells. Figure 46. Defining an assay with the Assay tool.
Microplate Manager User Guide Assay Info To rename your assays, annotate them, or change their colors, select Assay Info... from the ASSAY menu. This will open the Assay Information dialog box. Figure 47. Assay Information dialog box. Each assay will have its own tab. Click on the tab of the assay you want to change. Enter a new assay name and notes about the assay in the appropriate fields. Assays may have any name up to 30 characters in length. Notes are printed with the reports.
Chapter 5. Templates Defining Standards and Unknowns After you have defined the assays in the template, you are ready to identify the wells containing standards and unknowns on your template. Identifying the well types tells Microplate Manager how to use the well data from a reading in the resulting reports. You should define any assays before specifying well types. To format wells containing standards, click on the Format as Standards button, then click or drag on the standard wells in the template.
Microplate Manager User Guide unknown or standard shares the same number, they are part of the same replicate group (see page 68). Clicking on several unknown or standard wells in succession will number them sequentially. You can also number your wells sequentially using the Autofill buttons. Click on Autofill Left to Right and drag across multiple rows and columns of wells with your Standard or Unknown pointer. The wells will be numbered sequentially from left to right, then top to bottom.
Chapter 5. Templates If your well numbering is out of sequence and you want to correct it, select Resequence Well Names from the FORMAT menu. The wells will be renumbered in the correct order. This is a cosmetic change, and will not affect the analysis in any way. Defining Blanks and Controls Blank wells are used to measure background noise for accurate absorbance calculation. Positive control wells contain samples that define the upper limit of your data range.
Microplate Manager User Guide Replicate Groups A replicate group is a group of wells with the same standard or unknown. For each replicate group, a mean absorbance value (in the case of Endpoint or Multiple Plate Protocols) or velocity (in the case of Kinetic Protocols) is calculated, along with the standard deviation and coefficient of variation for the replicate group.
Chapter 5. Templates With your Standard or Unknown pointer, enter the number of replicates in each group in the field. Then click on the Autofill Left to Right or Autofill Top to Bottom button and drag across the group of rows and columns containing the sequential replicate groups. When you finish dragging, your replicate groups will appear, numbered sequentially. (Autofill Left to Right will number them sequentially left to right, then top to bottom.
Microplate Manager User Guide desired replicate group from the pulldown list of existing replicate groups within the dialog box. Entering Standard Concentrations If you have identified standard wells on your template (see page 65), you can enter the concentrations for those standards. Select Edit Standard Conc from the EDIT menu. This will open the Edit Standards dialog box. Figure 52. Edit Standards dialog box.
Chapter 5. Templates Click on the Enter Manually button to directly input the concentrations of your standards. Then type in the concentrations in the appropriate fields in the Conc. column. If a dilution series was created for the standards, the software can calculate the concentrations of each standard. Click on the Enter Automatically button, then click on the button that identifies the standard with the highest concentration and enter that concentration and the dilution factor in the appropriate fields.
Microplate Manager User Guide • Run at least three replicate wells for each dilution of standards and samples. • Run an adequate number of sample dilutions to ensure that more than one dilution lies in the middle of the curve. Running several dilutions will point out the limitations of the curve-fitting methodologies. Entering Dilutions of Unknowns You can enter dilution factors (if any) for the unknown wells in a template. Select Edit Unknown Dilutions... from the EDIT menu.
Chapter 5. Templates Click on the Enter Manually button to directly input the dilutions of your unknowns. Then type in the dilution factor in the appropriate fields in the Dilut. column. The number in the Dilut. column is the dilution factor, not the final concentration. For example, if the first unknown has a dilution factor of 1 (i.e., no dilution), and each unknown is twice as diluted as the previous unknown, enter 2 for the second unknown, 4 for the third unknown, 8 for the fourth unknown, etc.
Microplate Manager User Guide Figure 54. External Standards dialog box. If you are already using external standards, the External Standards checkbox will be selected. To stop using external standards, deselect the checkbox and click on OK. To begin using external standards, click on the External Standards checkbox. This will activate the File button. Click on the File button and a dialog box will open. Figure 55. Selecting the source file for the external standards.
Chapter 5. Templates Select the Data File containing the external standards and click on Open. The names of all assays with defined standards within the selected Data File will appear in the Source Assay pulldown menu. Figure 56. Selecting the source assay for your external standards. Under the Source Assay pulldown menu, select the assay that contains the desired external standards. When you are finished, click on OK to close the dialog and implement your changes.
Microplate Manager User Guide In the Unknown Concentration report, external standards are labeled XS1, XS2, XS3, etc. Saving Templates To save a template, select Save Data/Template or Save Data/Template As... from the FILE menu. This will open a standard Save As dialog box. Enter the name of the template and click on Save. The template will be saved with a .mpm extension. If you try to close the file before saving, you will be prompted to save the file before closing.
6. Data Files and Reports After you have run a Protocol, a Data File is generated and the resulting data is displayed automatically in one or more report formats, based on your selection in the Protocol dialog (see page 31). You can then save the Data File and organize your data in a variety of reports. Opening/Saving Data Files To save the data from a reading, with a Data File open, select Save Data/Template or Save Data/Template As... from the FILE menu. This will open a Save As dialog box.
Microplate Manager User Guide Limit, Matrix and Normalization Reports The Limit Report, Matrix Report, and Normalization Report allow you to set upper and lower absorbance or velocity limits on your data and display the results. Setting Data Limits To set data limits, first open the Limit, Matrix, or Normalization Report from the VIEW menu, then select Limits... from the OPTIONS menu. This will open the Limits dialog. Figure 57. Limits dialog.
Chapter 6. Data Formatting and Reports For Kinetic data, your limits will be adjusted velocities; for Endpoint data, your limits will be absorbances. Limit Report With the Data File open, select Limit Report from the VIEW menu to display this report, then set the limits by selecting Limits from the OPTIONS menu as described above.
Microplate Manager User Guide Figure 59. Matrix Report. The Matrix Report is similar to the Limit Report (see previous page), except that the data between the upper and lower limits are divided into 10 equal ranges, numbered 0–9. Each well with data within the limits is assigned a number, based on which range its data falls into. Normalization Report Select Normalization Report from the VIEW menu to display this report, then set the limits by selecting Limits from the OPTIONS menu as described above.
Chapter 6. Data Formatting and Reports Figure 60. Normalization Report. The Normalization Report displays absorbance or velocity data for each well as a percentage of a range defined by the limits. If custom limits are not selected in the Limits dialog box, the default upper limit is the highest standard, unknown, or control absorbance/velocity value in the assay, and the default lower limit is the lowest standard, unknown, or control absorbance/velocity value in the same assay.
Microplate Manager User Guide Figure 61. Control Report. The Control Report displays absorbance or velocity data for each well as a percentage of the range between the positive and negative controls in an assay. If no positive control is defined for the assay, the highest absorbance value or highest velocity among the wells defined as unknowns or standards is used. If no negative control is defined, then 0.000 is used as the negative control.
Chapter 6. Data Formatting and Reports Figure 62. Standard Curve Report. Features of the Standard Curve Display For replicate groups of standards, bars are plotted on the graph at each point corresponding to the standard deviation for the group. The Standard Curve Report displays curves for both internal and external standards. Internal standards are displayed in red, external in blue.
Microplate Manager User Guide If more than one assay has been defined on your plate, click on the tabs at the top of the window to switch between assays. Note that different assays can have different curves, using different regression methods, etc. Select the Show Labels checkbox to display labels for the individual standards on the curve. Select the Swap XY Axes checkbox to switch the X and Y axes of the curve. This will affect the curve display only, not the calculated data.
Chapter 6. Data Formatting and Reports The polynomial regression models (Linear, Quadratic, and Cubic) and logistic models are examples of global construction of a calibration function, while Cubic Spline and Point to Point are examples of a local approach. In general, when selecting a regression type for your data, you should start with the simplest curve fitting routine (i.e., linear) and then examine all four Transformation options (see below) for an acceptable curve fit.
Microplate Manager User Guide Figure 64. Selecting a transformation method. The equation at the bottom of the window will change based on your transformation selection. This selection will affect the calculated data. Axis Transformation You can change the axis display of the curve among four different combinations of linear and logarithmic: Linear(x)-Linear(y), Linear(x)-Log(y), Log(x)-Linear(y), and Log(x)-Log(y). Select from the Axis Transformation pulldown list.
Chapter 6. Data Formatting and Reports F statistic Significance Probability Figure 65. Analysis of Variance box. In the Analysis of Variance dialog box, the key statistics are listed in the last column. Within this last column, there are three subcolumns. From left to right, these subcolumns show the F statistic, the significance, and the probability that the difference between the two curves is significant.
Microplate Manager User Guide This report also provides statistics for each replicate group of wells, including mean, standard deviation, and coefficient of variation. With the Data File open, select Unknown Concentrations Report from the VIEW menu. Figure 66. Unknown Concentration Report. At the bottom of the window, the type of standard curve and its equation are shown.
Chapter 6. Data Formatting and Reports To change curve fit options or axis types, open the Standard Curve Report (see page 82) and select the desired regression method and axis type from the buttons in the dialog box. Reliability of Calculated Concentrations Results calculated from mean absorbance data outside the range of the standard curve may be unreliable.
Microplate Manager User Guide BSE Screening Report The BSE Screening Report is a Microsoft® Excel workbook with built-in calculation functions for reporting results from Bio-Rad's Platelia BSE Detection Kit. Microplate Manager can automatically generate this report using the data from a microplate reading.
Chapter 6. Data Formatting and Reports Figure 67. BSE Screening Report, generating using Custom Reports. The BSE Screening Report contains two worksheets: Analysis Settings and Report. Click on the navigation bar at the bottom of the workbook to display the different worksheets. The Analysis Settings worksheet contains settings for control validation and cutoff criteria.
Microplate Manager User Guide Figure 68. Analysis Settings in the BSE Screening Report. You can change the constants for k1, k2, and/or k3 to modify the cutoff criteria. These are the only settings that can be changed in the workbook. All other data fields and calculation functions are password-protected and unchangeable. The Report worksheet contains the BSE test data as recorded by the microplate reader. The results are displayed in a single sheet printable format.
Chapter 6. Data Formatting and Reports Each custom report template is released as a controlled document with its own part number and revision level. All validation procedures and results will be included with each release. The second section contains the validation results for the positive and negative controls, as specified in the Analysis Settings worksheet. Included in the control validation results is the number of valid controls and mean absorbance value for each control group (positive and negative).
Microplate Manager User Guide Each sample well shows the result (POS, NEG, or ???) and absorbance value. Wells flagged with a “*.***” are out of range, and blank wells are unformatted. Figure 72. Reading data. Raw Image Report (Ultramark only) The Raw Image Report displays an actual image of the microplate with the collected absorbance data. This report is only available if you have performed a plate reading with the Ultramark using a custom template (see page 25).
Chapter 6. Data Formatting and Reports Section of plate with absorbance data Figure 73. Example of a Raw Image Report (1536-well plate). Kinetic Protocols will create multiple images: one for each absorbance reading. Displaying the Image The IMAGE menu contains special tools for viewing your images.
Microplate Manager User Guide Figure 74. Image menu. The image is displayed in a window that can be resized by dragging the lower right corner of the window in and out. If Fit to Window is selected in the IMAGE menu, when you resize the window, the image will resize as well. If Fit to Window is deselected, the image will not resize when you resize the window, and scroll bars will appear if the image becomes too large to fit in the window.
Chapter 6. Data Formatting and Reports addition, if applicable, the assay number, well formatting, and current value of the well are displayed in the status bar. If you select Report Only Location from the IMAGE menu, the coordinates of the cursor and the absorbance value at that point will be displayed, but not the well information. Image Features In studying the features of a microplate image, it is useful to first magnify the image using the zoom tools.
Microplate Manager User Guide Show Pattern on the IMAGE menu displays the area of the well used for data collection (i.e., the extraction area). This too is specified in the Physical Dimensions dialog box (see below, and page 57). Show Translucent Pattern toggles the pattern display between transparent and opaque. Well and Pattern Color allows you to change the color used to display wells and patterns. Palette allows you to change the color of the entire image.
Chapter 6. Data Formatting and Reports Figure 76. Raw Data Report. For absorbance readings, the Raw Data Report will contain the absorbance value of each well in the microplate. The values are reported in ODs. If you elected to ratio the results of a dual wavelength reading in the Protocol (see page 26), the measurement wavelength reading is divided by the reference wavelength reading, and the resulting raw data will be displayed as simple ratios with no units.
Microplate Manager User Guide This report will be unavailable if you elected to ratio the results of a dual wavelength reading in the Protocol. In the Absorbance Report, the mean absorbance of all the blank wells in an assay is subtracted from the raw absorbance value of each non-blank well in the same assay. If no blank wells have been defined, the values in the Absorbance Report and the Raw Data Report will be the same.
Chapter 6. Data Formatting and Reports Figure 78. Velocity Report. The Velocity Report displays the velocities of your well readings, based on the analysis options you selected when you ran your Kinetic Protocol. Select Analysis Options from the OPTIONS menu to reopen the Analysis Options dialog box and change the settings. See page 40 for more information. Note that if you change the analysis options, the data will be automatically recalculated with the new settings.
Microplate Manager User Guide The Adjusted Velocity Report displays the adjusted velocity for each well calculated by subtracting the mean velocity of the blank wells from the raw velocities displayed in the Velocity Report. Kinetic Correlation Coefficient Report This report is available for Kinetic Protocol data. With the Data File open, select Kinetic Correlation Coefficient Report from the VIEW menu. Figure 80. Kinetic Correlation Coefficient Report.
Chapter 6. Data Formatting and Reports Figure 81. Kinetic Standard Errors Report. The Kinetic Standard Errors Report shows the calculated standard error of the velocity for each well. If you have large standard errors (more than 5–10% of the corresponding velocity), you should reset the limits (under OPTIONS > Analysis Options...) to focus on the linear portion of the results. Kinetic Plots Report This report is available for Kinetic Protocol data.
Microplate Manager User Guide Figure 82. Kinetic Plots Report. The Kinetic Plots Report displays plots of the changes in absorbance with time of all the wells in the microplate. The absorbance limits, total read time, time limits, and number of readings are listed below the chart. Kinetic Zoom Plot This report is available for Kinetic Protocol data. With the Data File open, select Kinetic Zoom Report from the VIEW menu.
Chapter 6. Data Formatting and Reports Figure 83. Kinetic Zoom Plot. This window shows additional velocity information for a single well. It displays a plot of the best linear curve fit and lists the calculated velocity, intercept, correlation coefficient, and standard error for the graph. Below the calculated values, the absorbance range and time range are listed. The data in the Kinetic Zoom Plot window is auto-scaled based on the data range for that particular well.
Microplate Manager User Guide Figure 84. Define Labels dialog box. You can specify up to six different labels. There are four predefined labels: Analyst, Chemistry, Plate #, and Source. Click on Add Predefined Labels to add these labels to your reports. To add some of these labels but not all, first add them all, then select the label(s) you don’t want and click on the Delete button. You can also add or insert user-defined labels. To append a label to the bottom of the current list, click on the Add...
Chapter 6. Data Formatting and Reports Fill Labels After you have created a label, you can enter the information to be included in that label immediately, if desired. Select Label Contents from the EDIT menu. This opens the Fill Labels dialog box. Figure 86. Fill Labels dialog box. In this dialog, each label name is followed by a text field. Enter the label information to be printed on each report. Click on OK when you are finished.
Microplate Manager User Guide Figure 87. Editing raw data. Your data will be displayed in a spreadsheet format. To change a particular number, select it and overwrite it. The edited data field will be permanently shaded in gray. You can highlight multiple number fields by dragging your cursor over the fields. You can highlight rows and columns by clicking on the button at the beginning of the row or column.
Chapter 6. Data Formatting and Reports With your data displayed, select Edit Outliers... from the EDIT menu. This will open the Select Outliers dialog box. Figure 88. Select Outliers dialog box. Click on the checkbox next to the well number and associated value that you want to flag as an outlier. This well will not be included in any reports, and will not be used to calculate the standard curve.
Microplate Manager User Guide Editing or Replacing the Template in a Data File After a Protocol has been run, the template from the Protocol File is automatically copied into the Data File and displayed (see Chapter 5 for more information about templates). When you save a data set, the template is saved with it. When you open an existing Data File, the template is always displayed with the data. This template may be edited or completely replaced by another template.
7. Printing and Exporting This chapter describes Microplate Manager’s functions for printing reports and exporting data. Printing All the reports may be printed to any printer supported by Windows®. To print a report when a report window is active, choose Print... from the EDIT menu. This will open the standard Windows® Print dialog box. Figure 89. Print dialog box. For additional settings such as page orientation, paper source, and resolution, click on the Properties... button.
Microplate Manager User Guide To automatically print reports immediately after a reading has been performed, click on the Reports... button in a Protocol dialog box. This will open a list of reports. Click on the appropriate checkbox(es) to print the desired report (s). When the Protocol is finished running, the selected report(s) will be printed. See page 31 for more information. To select the font that will be used to print the report, select Page Setup.. . from the FILE menu.
Chapter 7. Printing and Exporting To export, with your template or text-based data window open, select Export... from the FILE menu. From the Save as type: pull-down list, select the format you want to export in. Templates can be saved in .epc or .epf format. Data and reports can be saved as tab-delimited (.txt) or comma-delimited (.csv) values. There is a third export format (COL) for Kinetic data. In this format, all the readings are written to a file.
Microplate Manager User Guide Kinetic Data Formats COL Format The column format consists of (4 + (# of rows) * (# of cols)) lines. Line 1: “Col” (format signature) Line 2: # of readings (each in its own column) Line 3: #rows#cols (if this line is left empty, 128 is used) Line 4: timestamp1timestamp2...timestampN (timestamp format HH:MM:SS) Line 5: ... ... Line (col# + (4 + (row# - 1)*(# of cols))): ...
Chapter 7. Printing and Exporting Line ((reading# - 1)*(# of rows + 2) + row# + 4): ... ... Line ((reading# - 1)*(# of rows + 2) + row# + 4): ... Line ((reading# - 1)*(# of rows + 2) + row# + 5): #rows#cols (if this line is left empty, 128 is used) Line ((reading# - 1)*(# of rows + 2) + 4): timestamp2 Line ((reading# - 1)*(# of rows + 2) + row# + 4)): ...
8. Quick Guides Creating a New 96-Well Template See Chapter 5 for details. 1. Choose New Template > 12 x 8 from the FILE menu. 2. Click on the Define a new assay button on the template toolbar and drag on the template to define the location of each separate assay. 3. Use the formatting buttons on the template toolbar (Standard, Unknown, Positive Control, Negative Control, Blank) to define each well in each assay. Click on a button and drag in the defined assay to format the wells. 4.
Microplate Manager User Guide 9. a. Select Standard Curve Report or Unknown Concentration Report from the VIEW menu. b. Use the tabs to select the assay. c. Choose External Standards... from the OPTIONS menu. d. Choose source file for the external standards. e. Choose the source assay from within the source file. f. Repeat steps b–e for the other assays. g. Close the Standard Curve Report or Unknown Concentration Report. Choose Save Data/Template or Save Data/Template As...
Chapter 8. Quick Guides 5. Click Analysis Options to set the Kinetic analysis parameters. 6. Save the Protocol by choosing Save Protocol or Save Protocol As... from the FILE menu. 7. Initiate the Protocol by clicking the Run button. 8. At the end of the run, save the data by choosing Save Data/Template or Save Data/Template As... from the FILE menu. Creating and Running a New Kinetic Protocol with a New Template See Chapters 4 and 5 for details. 1. Select New Kinetic Protocol from the FILE menu.
Microplate Manager User Guide 6. Save the Protocol by choosing Save Protocol or Save Protocol As... from the FILE menu. 7. Initiate the Protocol by clicking on Run. 8. At the end of the run, save the data by choosing Save Data/Template or Save Data/Template As... from the FILE menu. Creating and Running a New Endpoint Protocol with a Stored Template See Chapter 4 for details. 120 1. Select New Endpoint Protocol from the FILE menu. 2. Fill in the information for the Protocol window: a.
Chapter 8. Quick Guides Creating and Running a New Endpoint Protocol with a New Template See Chapters 4 and 5 for details. 1. Select New Endpoint Protocol from the FILE menu. 2. Fill in the information for the Protocol window: 3. 4. a. Specify any initial wait. b. Specify any absorbance/ratio delay. c. Specify reading parameters. Click on Show Template to display a template of 96 undefined wells. a. Follow steps 2–8 of Creating a New 96-Well Template on page 117 to define the template. b.
Microplate Manager User Guide Creating and Running a Multiple Plate Protocol with a Stored Template See Chapter 4 for details. 122 1. Select New Multiple Plate Protocol from the FILE menu. 2. Fill in the information in the Multiple Plate Protocol dialog box: a. Specify any initial wait. b. Specify any absorbance/ratio delay. c. Specify reading parameters. d. Indicate if a plate loader is to be used. e.
Chapter 8. Quick Guides Creating and Running a New Multiple Plate Protocol with a New Template See Chapters 4 and 5 for details. 1. Select New Multiple Plate Protocol from the FILE menu. 2. Fill in the user-defined information for the Multiple Plate Protocol window: 3. Fill in the information in the Multiple Plate Protocol dialog box: 4. 4. a. Specify any initial wait. b. Specify any absorbance/ratio delay. c. Specify reading parameters. d. Indicate if a plate loader is to be used. e.
Microplate Manager User Guide 8. 124 At the end of the run, save the data by choosing Save Data/Template or Save Data/Template As... from the FILE menu.
Appendix A. Menus The pulldown menus in Microplate Manager change depending on the particular part of the application you are using. For example, if you are editing a template, one set of menu commands are available; if you are preparing a Protocols, another set of menu commands will become available. File This is the initial state of the FILE menu.
Microplate Manager User Guide Close closes the current file. All windows associated with the current file are closed. If the file has been modified, you will be prompted to save the changes. Save Data/Template saves the current data and/or template to a file. Save Data/Template As... opens a dialog box in which you can save the current data and/or template under a new file name or to a specific directory. Page Setup... opens a dialog box in which you can select a font for printing. Print...
Appendix A. Menus Edit Standard Conc... opens a form in which you can enter concentrations of standards. Edit Outliers... opens a dialog box in which you can exclude certain wells from all calculations. Set Assay... opens a dialog box in which you can change the assay to which the selected well belongs. Set Replicate Group... opens a dialog box in which you can change the replicate group of the selected well. Properties... displays the attributes of the selected well. Labels...
Microplate Manager User Guide Format This menu is available only when a template is active: Assay changes the pointer to a tool for defining the area of the microplate in which an individual assay is to be carried out. Select changes the pointer to a select tool. Blank–Undefined changes the pointer to tools for setting the well type. No Autofill turns off the autofilling functions.
Appendix A. Menus External Standards... opens a dialog box in which you can select another file from which to import standards to be used in calculating unknowns. When the Raw, Absorbance, or Unknown Concentration Report window is open, Limits... becomes unavailable. When one of the Kinetic report windows is open: Analysis Options... opens a dialog box in which you can specify time or absorbance limits for data collection. You also can choose the method of calculation of reaction velocities.
Appendix B. Glossary Assay: A set of wells grouped together for analysis. Blank: A well used for background subtraction. Endpoint analysis: Analysis based on a single absorbance value per well. Kinetic analysis: Analysis of the rate of change of absorbance for a well over a set period of time. OD (Optical Density): A measure of absorbance. Protocol: A group of settings that control a microplate reader. Includes incubator controls, filter settings, mix times, etc.
Appendix C. Ordering Information The following products can be ordered from Bio-Rad: Catalog Number Product Description 170-9520 Microplate Manager, Version 5.
Appendix D. Warranty Bio-Rad Laboratories warrants that the Microplate Manager software shall substantially conform, in all operational features, to Bio-Rad’s current specifications as published in Bio-Rad’s user and installation guides and that, when properly installed, it will be free of material defects which affect system performance.
Microplate Manager User Guide this manual and the software program contained herein may not be copied, in whole or in part, without the prior written consent of Bio-Rad, except in the normal use of the software or to make a backup copy. This exception does not allow copies to be made for others, whether or not sold, but all of the materials purchased (with all backup copies) may be sold, given or loaned to another person. Under the law, copying includes translating into another language or format.
Index Assays Annotating, 64 Defining, 62 Editing, 64 BSE Screening Report, 90 Computer and operating system requirements, 5 Concentrations Standards, 70 Custom Reports, 89 Data Editing, 107 Exporting, 112 Exporting to Excel, 113 Importing, 16 Kinetic formats, 114 Limits, 78 Data Files, 4 Changing templates, 110 Editing, 107 Identifying outliers, 108 Opening, 77 Saving, 77 Dilutions, 72 Dual wavelength readings, 27 Editing data, 107 Endpoint analysis, 35 Absorbance delay, 36 Ratio delay, 36 Examples, 14 Expo
Microplate Manager User Guide Exporting data, 46 Plate loading, 44 Ratio delay, 49 Saving data, 46 New features, 4 Outliers, identifying, 108 Page setup, 112 Physical dimensions of the microplate, specifying, 57 Plate images, 94 Plate loaders, 44 Preferences Selecting, 13 Print, 111 Preview, 112 Setup, 32 Protocol Files, 3 Protocols Common features, 23 Displaying templates in, 25 Endpoint, 35. See Also Endpoint analysis General, 21 Incubator controls, 28 Initial wait, 28 Kinetic, 38.
Index Scan Well command, 29 Scenarios, 16 Standard curves Comparing external and internal, 86 Displaying, 82 Standards, external Description, 75 Selecting in template, 73 Validating, 86 Variance analysis, 87 Starting Microplate Manager, 7 Status bar, 10 Templates Assays, 62.
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