Microplate Systems Microplate Manager 6 Software ® Instruction Manual | Version 6.
Microplate Manager 6 Bio-Rad Technical Support The Bio-Rad Technical Support Department in the United States is open Monday– Friday, 6:00 a.m. to 5:00 p.m., Pacific Standard Time. Worldwide technical support is available on the Web at http://www.consult.bio-rad.com/. Phone: (510) 741-6910, option 2, option 3 Fax: (510) 741-5802 E-mail: LSG.TechServ.US@Bio-Rad.com (U.S.) LSG.TechServ.Intl@Bio-Rad.com (International) Web: i http://www.consult.bio-rad.
Introduction Table of Contents Table of Contents........................................................... ii Introduction .................................................................... 1 Microplate Manager 6 Overview............................................................1 Menu Bars and Icons.............................................................................2 File Menu .................................................................................................. 3 Window Menu ....
Microplate Manager 6 Exporting Data ........................................................................................ 18 Exporting Reports ................................................................................... 19 Exporting Custom Screening Reports (BSE, TSE, TSE, NSP) .............. 20 Procedure for Custom Export Reports.................................................... 20 BSE Custom Screening Report ..............................................................
Introduction Running Kinetic Assays .............................................. 52 Reading a Kinetic Assay......................................................................52 Analyzing Kinetics Data.......................................................................53 Kinetic Zoom Plots .................................................................................. 54 Viewing Raw Data at Kinetic Time Points............................................... 55 Defining Kinetic Parameters .........
Microplate Manager 6 3 Selecting Curve Fit and Customizing Curve Fit Plot........................73 4 Entering Experimental Info ...............................................................77 5 Setting up the Report........................................................................78 Percent Control Assay Tutorial .................................. 82 1 Defining the Template ......................................................................82 2 Setting up the Equations .........................
Introduction Introduction This manual assumes that you are familiar with the standard commands and functions associated with the Windows® or Mac® operating system, such as opening, closing, and saving files, and moving and clicking your mouse. Some of the features and functions in Microplate Manger 6 will be slightly different depending on which microplate reader you are using (xMark or iMark). These differences are described in the text.
Introduction Microplate Manager 6 can perform four types of microplate readings. • Endpoint Reads are used to acquire a single absorbance reading from each well. • Kinetics Reads are used to acquire a series of absorbance readings from each well over a user-defined time interval to calculate reaction rate etc. • Plate 2-1 Reads are endpoint data from two plates with data from the second plate subtracted from the data from the first plate.
Introduction File Menu After creating a New Experiment or opening an existing data file, the File Menu will list the main program functions. The nine icons grouped at the left end of the Main Toolbar correspond to the same function items listed under the File Menu.
Introduction • Export Data to Excel • Read a Plate • Print Report/active window • Print Preview of Report Window Menu After creating a New Experiment or opening an existing data file, the Window Menu will list the display functions for raw data, results and reports. The six icons grouped at the right end of the Main Toolbar correspond to the same function items listed under the Window Menu. Full Window menu descriptions are below: 4 • Open the Info Window to enter experiment information.
Installation and Instrument Setup Installation and Instrument Setup Minimum Requirements Computer • Windows XP, Windows Vista or Mac OS 10.
Installation and Instrument Setup For Mac Users: 1. Insert the software CD into the CD-ROM drive. The installer user interface displays. 2. Drag the Microplate Manager 6 icon from the CD folder to the Applications folder on your computer hard drive. iMark Reader Instrument Setup 1. Turn the iMark reader on. 2. Enter a five-digit password on the iMark keypad to enable remote control for iMark. 3. Select New Experiment from the File menu.
Installation and Instrument Setup ¾ If filters are added, removed, or their positions changed, the changes must be entered on the iMark reader keypad, so that filters are displayed properly in MPM 6. Entering Filters On the iMark Keypad: 1. Click the Edit button. 2. Press down arrow button 2 times to select Filters, and press the Enter button. 3. Using the keypad, select numbers by using arrow buttons to move through positions 1-8. 4. Press the Enter button to save settings. 5.
Installation and Instrument Setup xMark Instrument Setup 8 1. After opening MPM 6 for the first time, choose New Experiment from the File menu. 2. Select xMark from Select Reader. 3. Select the Reader tab. In the Wavelength area, set up spectral values from 200.0 – 1000.0 nm. 4. Set up temperature parameters in the Incubator tab, if the incubator is required.
Installation and Instrument Setup 5. Select the Shaker Only tab to set up mixing parameters (xMark only).
Quick Start Quick Start Setting Up an Experiment 10 1. Launch software from the desktop shortcut. 2. Click on the New Experiment icon or choose File > New Experiment to set up a New Experiment. 3. The program title bar displays “No Data - Protocol : Untitled”. 4. Select the available instrument under Select Reader. 5. Choose Plate Size (xMark only). The default is 96.
Quick Start Open a saved protocol or set up a new protocol (by setting up a template, and selecting the instrument, assay info, analysis options and print options). Refer to the Instrument Controls 1. Click File > Read New Plate. The Instrument Controls dialog displays. 2. Under the Reader tab, set the Reading mode: Endpoint, Kinetic, or Plate 2-1. If you are using an xMark, Spectra is another Reading mode option. 3.
Quick Start Incubator Controls Under the Instrument Control dialog box, select the Incubator tab (xMark only) 1. Click the Set to On button. 2. Enter a Temperature Set Point. 3. Click the Monitor Temperature button if you want to monitor the temperature continuously. You can also activate the Temperature monitor from the File menu. This window regularly monitors the xMark temperature status, listing the current temperature & set temperature and whether the incubator is on or off.
Quick Start Reading Microplates After all Instrument settings have been completed, press the Start Read button to read the plate. Data analysis results can be viewed, changed, saved, and printed after raw data are received from the instrument. See the Analyzing Data Using a Standard Curve and the Analyzing Data Using Cutoffs sections.
Data and Protocol Files Data and Protocol Files The following table explains how protocol and data files differ, and describes the file types MPM 6 uses to save each one. Protocol Data .pro is the extension for a protocol file. .mpl is the extension for a data file. A protocol file includes all the settings for reading a plate (measurement wavelength, shake, runtime, ...
Data and Protocol Files The Sample Data files are stored in the following default location: C: \ Bio-Rad \ Microplate Manager 6 \ Sample Data The Sample Protocol files are stored in the default location C: \ Bio-Rad \ Microplate Manager 6 \ Sample Protocols Newly-generated Protocol and Data files are stored in the default location: C: \ Bio-Rad \ Microplate Manager 6 For Macintosh Computers: The Data, Protocols, Sample Data & Sample Protocols folders are all installed in the program application folder on t
Data and Protocol Files Data AutoSave When the AutoSave feature is selected in the Instrument Setup Window, the data will be saved automatically after each read (and will not be displayed). The file name will automatically have the date/time and number of the read. This file name can be changed later. Importing and Exporting Data Microplate Manger can import or export data to other programs which accept.xls, .csv, or .txt file types.
Data and Protocol Files 3. Change file Extension from ".epc " to " .csv " In Microplate Manager 6, 1. Choose New Experiment. 2. Select File > Import Data 3. Select File > Excel file type (*.xls,*.csv,*.txt )“ , and click Open. 4. In the Import Setup dialog, click on Matrix. 5. Enter the # Rows preceding data. 6. Enter the # Columns preceding data. Click OK. 7. The Raw Data displays. Save file in Microplate Manager 6. Importing Data from Excel MPM 6 will import Excel files using *.txt, *.
Data and Protocol Files Single-Click on the first well of the range of data and paste. The entire range of data will be pasted into the matrix. (Do not Doubleclick on the well for pasting data.) Data can also be entered into the program by typing from the keyboard. Exporting Data or Reports Both raw data and result data can be exported to Excel from Microplate Manager 6. Exporting Data 18 1. Click on the Export Data icon on the main toolbar and name the file to be exported.
Data and Protocol Files The exported file will be saved as an Excel (.xls) file. Exporting Reports 1. To export data in a report format, including table or matrix headings, select the desired items such as Info, Plate Data, Standard List, Result List, etc. from the list in the Content Selector window under the Edit menu. 2. Choose File > Export Report, name and save the report.
Setting Up a Protocol Exporting Custom Screening Reports (BSE, TSE, TSE, NSP) (Windows PC only, using Bio-Rad custom Excel templates BSE, TSE, TSE(NSP)…) The Custom Export feature under File menu is for automatically exporting data and generating a Screening Report for a series of Bio-Rad Platelia BSE and related detection kits. This Custom Export feature is used together with the BSE (and related) Protocols in the Sample Protocol folder and the “Custom Templates” folder in the Microplate Manager 6.
Setting up a Protocol 5. 6. Select File >Custom Export. Select desired Template (which contains the built-in calculation functions for reporting the results for this kit). 7. Once the Template is selected, the program will automatically open Excel and display the correct Screening Report. The BSE (or other) Screening Report will be saved automatically with the name that was entered. BSE Custom Screening Report This BSE (or other) Screening Report contains two worksheets; Analysis Settings and Report.
Setting Up a Protocol Section, the Control Validation Section, the Cutoff Values Section, and the Matrix Data including the Sample info. In the Information section, the report template, technician, plate, reader, and reading parameters are given. The custom report template is uniquely identified with a name, part number and revision number. Each custom report template is released as a controlled document with its own part number and revision level.
Setting up a Protocol The matrix of the 96-well absorbance data, including the well identifiers, is shown in the fourth section. Positive control wells (PC1 and PC2) and negative control wells (NC1 through NC4) and their absorbance values are shown. Controls that do not pass validation are flagged in red with strikethrough marks. Each sample well shows the result (POS, NEG or ???) and its absorbance value. Wells flagged with a *.*** are out of range, and blank wells are unformatted.
Setting Up a Protocol Setting up a Protocol Setting up protocols automates your test processes. Parameters for frequently-run assays can be set up and saved once, and used whenever the assay is performed. To set up a assay protocol, follow these steps: 1. Set up a Template 2. Select Instrument Setup settings 3. Select Instrument Controls 4. Enter Assay Info 5. Select an Analysis Option 6. Select Report Options 7.
Setting up a Protocol Setting Up A Template The template defines the identifier locations for your plate data. A template must always be present in order to see any Result Data or Report window. The template controls allow you to edit your template, as well as to enter equations and dilutions. The controls are available as menu items, or as icons under the Template Window toolbar. Click on the Template icon in the Main toolbar or choose Template under the Window menu to open a template.
Setting Up a Protocol To highlight the entire plate, choose Select All under the Edit menu. To clear the template, press the delete key. To re-size Template columns, in order to display long ID names, position the cursor in the column number at the top of the window and drag it to the right. To AutoFill a series of identifiers that begin with a number, highlight the area to be filled in, click on fill a series icon. In the Fill Series window, enter the desired concentration or dilution factor.
Setting up a Protocol Entering Dilutions Dilutions can be entered for any sample well (except for blanks or standards). To enter Dilutions: 1. Click the Template View icon. 2. Select Dilutions, or choose View > Dilutions in the Main menu. In the Dilution window, the default is all the wells filled in with 1.0, indicating that the dilution is one (no dilution). are The dilution factor is the ratio of 1 to the value entered. For 1:2 dilution, enter the dilution factor as 2.
Setting Up a Protocol Select up to 6 Custom Labels from a pop-down menu by selecting one of the items in the list, or by entering a new Label ID in the edit field. The selected Labels will appear in the Info Window and will be saved in a Protocol. Custom Labels can also be set up and used for Custom BSE (or other) Screening Reports. Select and Customize Curve Fit Plot 28 1. Click Curve Fit icon to select desired Curve fit. 2. Click on the Customize Plot icon.
Setting up a Protocol 3. In the Curve Fit Plot Editor, select Auto/ Manual Scaling, Number Format, Axes labels, etc. for Curve Fit Plot.
Setting Up a Protocol Setting up the Report Contents 1. Click Report Icon in Main Toolbar. 2. Click the Select Report Contents icon, and then, select the Select Table Columns icon from the Results toolbar. 30 3. Select contents (Raw Data, Results Data, Plot, Info, Samples Table…) to include in the report by moving items up to the top section of the list and arranging them in the desired order. 4. Select columns (Standard #, Well ID, Concentration, Absorbance, etc.
Setting up a Protocol 5. Click Format Report Editor icon in the Report toolbar to format the report with titles, number format, headers and footers. Saving the Protocol 1. Choose File > Save Protocol. 2. Name the protocol and click Save. .
Analyzing Data Using a Standard Curve Analyzing Data Using a Standard Curve The standard curve is a plot of the concentration of the standards you have designated in the template (on the x axis) versus the readings from the instrument (in OD units for an endpoint experiment (on the y axis).
Analyzing Data Using a Standard Curve Standard Curve Analysis Options Linear Fit For a linear fit, the best straight line is fitted to the data points, OD's (response) (yaxis) vs. the standard concentration (x-axis). The equation of the line has the form y = Intercept + Slope * Conc The calculated intercept, the slope, the correlation coefficient (r) and chi2 values of the line are shown below the plot.
Analyzing Data Using a Standard Curve Zero-intercept Linear In the Zero-intercept of analysis a linear fit is used, but the intercept is set to Zero. This method of analysis is useful for assays which have a linear response to the concentration such as protein assays or other assays.
Analyzing Data Using a Standard Curve Semi-log Fit When Semi-Log is selected in the Curve Fit dialog box, a plot of the response (y axis) vs. the logarithm of the concentration (x-axis) is obtained as shown below: For the semi-log fit, the equation is: y = Slope*Log10(conc)+ Intercept The calculated intercept, slope, the correlation coefficient (r) and chi2 values of the line are shown below the plot.
Analyzing Data Using a Standard Curve Log/log Fit The Log/Log curve fit option fits the best straight line to the data consisting of the logarithm of the OD (response) vs. the logarithm of the concentration. For the Log/log method, the equation for the standard curve is: Log10(y) =Intercept + Slope*Log10(conc) In the plot, the concentration and the OD axes are displayed on the log scale.
Analyzing Data Using a Standard Curve Log/Logit and 4-Parameter Fit Both the Log/Logit and the 4-Parameter curve fit methods are based on the same equation: A-D y = ______________ + D 1 + ( conc / C )B which can be expressed in the equivalent form: logit y = a + b * log10(conc) where logit y = ln (y'/1-y'), B = -b/ln10 = slope at inflection point of curve y' = (y-D)/(A-D) C = EXP(a/B) = concentration at midpoint between A & D The difference between the Log/Logit and the 4-parameter curve fit options is i
Analyzing Data Using a Standard Curve The 4-parameter method is usually preferred over the Log/Logit method, because the results for the 4-parameter method are more accurate. In both the Log/Logit and the 4-Parameter plots, the concentration axes are displayed on the log scale. The parameters A, B, C, D, the chi2 values of the standard curve are shown below the plot.
Analyzing Data Using a Standard Curve Point to Point In the Point to Point method of analysis, the data points are simply connected. No fitting of the data points to a line or curve is done. For reactions where there is a lag time or a plateau, it is often convenient to look at the data using this plot in order to see the true linear range of the assay. Cubic Spline Cubic spline is a piecewise polynomial approximation in which a set of data points are joined by a series of curve fits.
Analyzing Data Using a Standard Curve A minimum number of standards that pass certain criteria are required for each type of curve fit. For the best results, always use more than the minimum number of standards required to do the calculation. If these requirements are not met, the Fitting will Fail. Interpolation Using a Previous Standard Curve It is frequently convenient to run one plate containing all the standards for a standard curve and other plates having only samples.
Analyzing Data Using a Standard Curve To obtain a printed standard table report for the imported standard curve, print the imported standard curve file separately. Customizing Standard Curve Plots To customize the standard curve plot with axes labels or different X, Y ranges: 1. Click on the Customize Plot icon. 2. In the Plot Editor window, select the Plot Tab, to customize the color of the lines or point size and choose grid line on or off.
Analyzing Data Using a Standard Curve Equations and Calculations Definitions of the statistical parameters calculated for a set of values are discussed below. For data sets with dilutions, the equations used in calculating the weighted average of dilutions are also described. • Mean • Standard Deviation, SD • % Coefficient of Variation, % CV • Root Mean Square Error, RMS • Chi-square statistic value • Correlation Coefficient, r • Weighted Average of dilutions, Wt.
Analyzing Data Using a Standard Curve A residual is the difference between an observed value of the response variable and the value predicted by the regression line. That is, Residual = observed y - predicted y. The chi square value is a measure of how close the observed values are to the calculated values. A chi value of zero means that the observed values are equal to the calculated values. Likewise, small chi values indicate a good fit.
Analyzing Data Using a Standard Curve Error of the interpolated concentration (concy) is related to the error of the response sy according to s conc = ( δ conc(y)/ δ y) sy The estimated standard error of concentration (SEMconc) calculated from the mean response of n replicates is SEMconc = (δconc(y)/δy) RMS(y) / SQR n The (δconc(y)/δy) depends on the form of the standard curve.
Analyzing Data Using Cutoffs Analyzing Data Using Cutoffs The Cutoff Result analysis options are used for assays including one or two controls (and no standard curve). All the samples are compared to the cutoffs or controls. To set up a Cutoff Analysis Report: 1. Click on the Result icon on the main toolbar. 2. Click on the Cutoff Report icon on the Result Window.
Analyzing Data Using Cutoffs The three cutoff display options in the menu are described here. Either two or four cutoffs are used based on the user definitions in the Cutoffs tab window. • Gray Scale: In the Gray Scale window, the data values are shown numerically and in shades of gray. Results equal to or below the lowest cutoff are white, results equal to or above the highest cutoff are black, and results between the two cutoffs are shown in shades of gray.
Analyzing Data Using Cutoffs Reference can be made to standard or sample IDs that have been defined in the Template setup • Standards: • Unknowns: enter identifier • Blanks: enter ! followed by concentration enter # In all Cutoff equations, Microplate Manager 6.0 uses the averaged value of that group. Examples of valid expressions: • U01 • (U01+.
Analyzing Data Using Cutoffs Data transformed – viewed as a Percent (0 - 100 %) • Low Cutoff: 0 • High Cutoff: 100 Using values from a Standard Curve – standards 0, 5, 10, 25, 50, 100, 250, and 500 defined on template • Low Cutoff: ! 0 • High Cutoff: ! 500 Using an equation – sample (SAMPLE2) well(s) defined on template • Low Cutoff: 0 • High Cutoff: (SAMPLE2-0.025)*100 Setting Up a Percent Control Cutoff Analysis Report Define the desired cutoffs in the Cutoff Report Editor window.
Analyzing Data Using Cutoffs The Control wells are defined on the template. The resulting Extended Cutoff Report, is shown here with the high values shown in red, the low values in blue and the intermediate values in white: 1. To view the results in tabular format, click on the Report icon in the main toolbar.
Analyzing Data Using Cutoffs 2. Click the Select Table Columns icon in the Report Toolbar.. 3. In the Columns Selector window, move Ext. Cutoff Value into the included item. Move the items up or down to select which items to include in the Report and change their order. Select Sorted By Mean and Descending order.
Analyzing Data Using Cutoffs The information entered in the Info window is automatically listed at the top of the Report. Concentration unit information is displayed on the Tables and Curve fit Plot.
Running Kinetic Assays Running Kinetic Assays Running assays kinetically has many advantages: • Kinetic assays are more accurate. The errors in endpoint assays such as the time interval between substrate addition and reading in wells across the plate, differences in the optical uniformity of wells, and the substrate volume in the wells are eliminated. • Using kinetics, assay sensitivity is greater for competitive assays.
Running Kinetic Assays 3. Enter the interval between the start of two consecutive reads in the Interval column. The interval time is the time necessary for one read plus mix time specified. 4. Enter mix time, if desired. In the above kinetic read example, the reader will do 40 readings with 30 second intervals and no mix. 5. After the parameters are set, click the Read Plate icon to start the readings.
Running Kinetic Assays Kinetic Zoom Plots Zoom in on a range of wells or a single well in the Kinetic Plots display to get an enlarged view of the plots. To zoom on a single well: • Right-click on the desired well. To return to the display of the whole plate: • Right-click in the plot and click on zoom out. To zoom in on a range of wells: • 54 Shift-drag over the desired range.
Running Kinetic Assays To zoom in on a single well or return to the display of the whole plate: 1. Right-click in the desired plot. 2. Select Zoom In to see large view or Zoom Out to return to the whole plate. To change the plot properties, such as axes labels and scaling, click the Customize Plot button. Viewing Raw Data at Kinetic Time Points To see raw data values, go to the View menu and uncheck Show Kinetics to return to the Raw Data matrix.
Running Kinetic Assays After the read is completed, data can be excluded from calculation by defining a new time range or new OD range. Original data is not lost, but is not included in the current calculation. Defining Kinetic Parameters Several alternative kinetic parameters can be used for analyzing the raw kinetics data in the program: • Rate - The kinetic rate (slope of the OD vs. time curve) will be calculated by linear regression, using all the data points within the selected OD and time range.
Producing Reports Running Spectra Assays (xMark only) 1. Select File > Instrument Setup, Spectra Reading Mode. 2. Enter desired wavelength range and increment interval. 3. Enter desired starting and ending Well Range values. 4. Click Start Read. 5. Click the Raw Data icon on the main toolbar. 6. Click the Show Plots icon or select Show Spectra under the View menu to view the Spectra plots.
Producing Reports Producing Reports A template must always be present in order to see any Result Data or Report Tables. Setting Up Reports To create a Report, click on the Report icon in the main toolbar, or select Report under the Window menu. The Edit and the View menus include the options shown below: A template must always be present, in order to see any Results Data or Data in the Table. Click the Report icon on the main toolbar.
Producing Reports Click the Select Report Contents icon in the Report toolbar. Select Report Contents Select Table Columns Format Report Print Preview In the Contents Selector dialog, move the contents items above or below the “Include Items Above” line to select items and arrange the order. 1. Click Report icon in Main toolbar. 2. Click Select Report Contents icon in the Report toolbar. 3. Click the Select Table Columns icon in the Report toolbar. 4.
Producing Reports 5. Select sorting option, Ascending or Descending Separate tabs for Standard and Samples. 6. Click the Format Report icon in the Report toolbar. In the Report Format Editor window: 1. Check On, User-defined response name. 2. Enter % B/Bo in Name Edit field. A section of the Report Standard and Samples Tables with user-defined name ‘%B/Bo” as a column header is shown here.
Producing Reports With Report selected under the Window menu, choose File > Print to print the report. Printing Reports 1. Choose File > Page Setup, to set margins and paper size and orientation. 2. With Report selected under Window menu & Report as the active window, Choose File > Print to print the report.
Producing Reports Or, click on the Report icon in the Main toolbar. 62 3. After setting up the report, click on Print Preview icon to preview the entire report. 4. Click on Print icon to print the Report.
Appendix Customizing Reports 1. Click on the Report icon on the main toolbar. 2. Click on the Format Report icon on the report window. In the Report Format Editor window, the mean response column heading in the Report can be customized, for Results that have been transformed by equations. For example, the heading %B/Bo entered here will appear in the report column heading as “Mean %B/Bo”. In this window, the Number Format for the report is selected.
Appendix Appendix References [1] Channing Rodgers, R. P. (1984) "Data Analysis and Quality Control of Assays: A Practical Primer" in Practical Immunoassay W. R. Butt (ed), Marcel Dekker, NY, 253-308. [2] Press, William H.; Flannery, Brian P.; Teukolski, Saul A.; Vetterling, William T. “Numerical Recipes in C: The Art of Scientific Computing”, Cambridge Univ. Press, NY, 1988. [3] Davis, S.E., Munson, P.J., Jaffe, M.L. and Rodbard, D. (1980), J. of Immunoassay, 1, 15. Radioimmunoassay Data Processing.
Appendix Quantitative ELISA Assay Tutorial A tutorial showing how to set up an ELISA Assay, including a standard curve and equations to transform the absorbance data into percentage values of the maximum bound well, is given here. 1. To set up the protocol, Choose File > Instrument Setup. 2. Select Endpoint Reading Mode and the desired wavelength. 3. Open sample data file (Percent Max Binding Assay) with its protocol & template.
Appendix Setting up the Equations 1. Click Template View icon, select Equation from popup. 2. Click Equation Editor icon. Choose % Max from Equation Name popup to view equations to be applied sequentially to data.
Appendix Selecting and Customizing a Curve Fit Plot 1. Click Curve Fit Plot icon in Main Toolbar. 2. Choose Fitting Function icon in Curve Fit Plot toolbar. 3. Select 4-Parameters from list. 4. Click Customize Plot icon in Curve Fit Plot toolbar. In the Curve Fit Plot Editor window: X-axis tab: enter: Concentration Y-axis tab: enter: % B / Bo in the Axes label edit fields Entering Experimental info 1. Click on the Info icon on the Main Toolbar. 2.
Appendix Dilution Data Assay Tutorial 1. Defining the Template 2. Setting up the Dilutions 3. Selecting Curve Fit and Customizing Curve Fit Plot 4. Entering Experimental Info 5. Setting up the Report An example of how to set up an ELISA Assay including a dilutions and a standard curve is given here. The Report is customized to show interpolated concentration, dilution factor, conc X dil factor, and mean concentration for the samples. Open sample data file (Dilution Data.
Appendix 5. Enter as below and click OK 6. 7. 8. Type 0 in well H1 & H2 and press Enter. Type S01 in well A3 and press Enter. Highlight area well A3-H12. (Click & hold mouse on well A3, drag mouse to well H12, and release.) 9. Click on the Fill Series icon in the Template Toolbar 10.
Appendix 11. Below is the finished template o Standards are defined 200, 100, 50, 25, 12.5, 6.25, 3.125, & 0. o S01 to S20 are all of the unknown samples to be analyzed. 2 Setting up the Dilutions 70 1. Click Template View icon in the Template Toolbar 2.
Appendix 3. Highlight area well A3-D3. (Click & hold mouse on well A3, drag mouse to well D3, and release.) 4. Click on the Fill Series icon in the Template Toolbar 5. Enter as below and click OK 6. Dilutions are filled in automatically by the Fill Series 7. Click on the Copy icon in the Template Toolbar 8.
Appendix 9. Click on the Paste icon in the Template Toolbar 10. E3 to H3 are filled 11. Highlight area well A3-H12. (Click & hold mouse on well A3, drag mouse to well H12, and release.) 12. Click on the Fill Right icon in the Template Toolbar 13.
Appendix 3 Selecting Curve Fit and Customizing Curve Fit Plot 1. Click Curve Fit Plot icon on the Main Toolbar 2. Click Fitting Function icon in Curve Fit Plot Toolbar 3. Select 4-Parameters from list 4.
Appendix 5. 74 In the Curve Fit Plot Editor window under Plot tab, select the Color, Symbol, and Line style for the Data Points and Curve Fit Line.
Appendix 6. In the Curve Fit Plot Editor window under X-axis tab, enter Concentration for the axis label. Set the Range to Auto. 7. In the Curve Fit Plot Editor window under Y-axis tab, enter Absorbance for the axis label. Set the Range to Auto.
Appendix 8.
Appendix 4 Entering Experimental Info 1. Click on the Info icon on the Main Toolbar 2. Enter Title of assay and any experimental Comments. 3. Enter concentration unit (ng/ml) in the Info window. (This unit will appear in the Conc column header of the Report Tables.
Appendix 5 Setting up the Report 78 1. Click Report icon in Main Toolbar 2. Click Select Report Contents icon in the Report Toolbar 3. Move the items up or down to select which items to include in the Report and change their order. Select Dilution Factor to list the template dilutions in the Report. Select Fitting Info to list the fitting parameters in the Report. 4.
Appendix 5. Select Columns to be included for Standard and Sample Tables in Report window. Move the items up or down to select which items to include in the Report and change their order. Select Sorted By Std # and Descending order for Standards. Select Sorted By Sample ID and Ascending order for Samples.
Appendix A section of the Data Analysis Report for Standards and Samples Tables with Average Concentration is shown here.
Appendix 6. Click the Print Preview icon in the Report 7.
Appendix Percent Control Assay Tutorial 1 Defining the Template 2 Setting up the Equations 3 Setting up the Screening Report 4 Entering Experimental Info 5 Setting up the Report An example of how to set up an ELISA Assay that uses a control and equations to transform the absorbance data into percentage values of the control well (CNTRL) is given here. This assay has no standards and no standard curve. Open sample data file (Percent Control.mpl) with its protocol and template.
Appendix • CNTRL is for the control wells. • U01 to U29 are all of the unknown samples to be analyzed. 2 Setting up the Equations 1. Click Equation Editor icon in the Template Toolbar 2.
Appendix 3. Click Template View icon in the Template Toolbar. 4. Select Equation from popup. 5. To apply this equation to the data, right click the mouse and select the % CNTRL equation from the popup. 3 Setting up the Screening Report 84 1. Click on the Result Data icon in the Main Toolbar 2.
Appendix 3. Define the desired cutoffs in the Cutoffs Report Editor window. Well identifiers used in the Cutoff Report Editor must be first defined on the template. User defined cutoffs are used in this data file. • The Max High (++) is defined as ≥ 85% of the control (CNTRL) for all samples. • The High (POS) is defined as ≥ 75% of the control (CNTRL) for all samples. • The Low (NEG) is defined as ≤ 15% result value for all samples.
Appendix 86 4. Click the Report View icon in the Result Toolbar 5. Select Extended Cutoff Report from popup 6.
Appendix 4 Entering Experimental info 1. Click on the Info icon on the Main Toolbar 2. Enter Title of assay and any experimental Comments. 3. Enter concentration unit (ug/ml) in the Info window. (This unit will appear in the conc column header of the Report Tables.
Appendix 5 Setting up Report 88 1. Click Report icon in Main Toolbar 2. Click Select Report Contents icon in the Report Toolbar. 3. Move the items up or down to select which items to include in the Report and change their order. 4. Click the Select Table Columns icon in the Report Toolbar.
Appendix 5. Select Columns to be included for Sample Table in Report window. Move the items up or down to select which items to include in the Report and change their order. Select Sorted By Mean and Descending order. 6. Click the Format Report icon in the Report Toolbar. 7.
Appendix A section of the Data Analysis Report for Samples with user-defined name % CNTRL as a column header is shown here. 8.
Appendix 9.
Appendix Percent Max Binding Assay Tutorial 1 Defining the Template 2 Setting up the Equations 3 Selecting Curve Fit and Customizing Curve Fit Plot 4 Entering Experimental Info 5 Setting up the Report An example of how to set up an ELISA Assay including a standard curve and equations to transform the absorbance data into percentage values of the maximum bound well is given here. Open sample data file (Percent Max Binding.
Appendix • Blanks are defined as #. The average of these # wells will be subtracted from all the wells. • NSB are non-specific binding wells. • MAX is for the maximum binding well (i.e. the Bo well). • S01 to S21 are all of the unknown samples to be analyzed. 2 Setting up the Equations 1. Click Equation Editor icon in the Template Toolbar. 2.
Appendix 3. Click Template View icon in the Template Toolbar 4. Select Equation from popup 5. To apply this equation to the data, right click the mouse and select the % MAX equation from the popup. 3 Selecting Curve Fit and Customizing Curve Fit Plot 94 1. Click Curve Fit Plot icon on the Main Toolbar 2.
Appendix 3. Select 4-Parameters from list 4. Click Customize Plot icon in Curve Fit Plot Toolbar 5. In the Curve Fit Plot Editor window under Plot tab, select the Color, Symbol, and Line style for the Data Points and Curve Fit Line.
Appendix 6. 96 In the Curve Fit Plot Editor window under X-axis tab, enter Concentration for the axis label. Set the Range to Manual with the minimum 1.0 and the maximum 100000.
Appendix 7. In the Curve Fit Plot Editor window under Y-axis tab, enter % B / Bo for the axis label. Set the range to Manual with the minimum 40.0 and the maximum 100.00.
Appendix 8. 98 The customized curve fit plot shows the curve fit parameters below the curve.
Appendix 4 Entering Experimental Info 1. Click on the Info icon on the Main Toolbar 2. Enter Title of assay and any experimental Comments. 3. Enter concentration unit (ng/ml) in the Info window. (This unit will appear in the conc column header of the Report Tables.
Appendix 5 Setting up the Report 100 1. Click Report icon in Main Toolbar 2. Click Select Report Contents icon in the Report Toolbar. 3. Move the items up or down to select which items to include in the Report and change their order. Select Fitting Info to list the fitting parameters in the Report. 4.
Appendix 5. Select Columns to be included for Standard and Sample Tables in Report window. Move the items up or down to select which items to include in the Report and change their order. Select Sorted By Std # and Ascending order for Standards. Select Sorted By Mean and Descending order for Samples. 6. Click the Format Report icon in the Report Toolbar 7.
Appendix A section of the Data Analysis Report for Standards and Samples Tables with userdefined name % B/Bo as a column header is shown here.
Appendix 8. Click the Print Preview icon in the Report Toolbar 9.
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