Manual

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IMPORTANT! Pay close attention to vortexing, shaking, and incubation instructions.

Deviation from the protocol may result in low assay signal and assay variability.

For use with Instruction Manual #

Bio-Plex Pro Human Isotyping Assays 10028370

This guide can be used to prepare and run a full 1 x 96-well assay plate.

For more information on a given step, refer to the complete instruction

manual. New users can download the manual, which includes detailed

instructions and a list of kit components, at www.bio-rad.com/bio-plex.

Initial Preparation

1. Plan the plate layout.

2. Start up/warm up the Bio-Plex

system (30 min).

Bring the 10x wash buffer, assay buffer, and isotyping diluent to room

temperature (RT). Keep other items on ice until needed

Begin to thaw frozen samples

3. Prime wash station for ﬂat bottom plate or set vacuum manifold to

–1 to –3" Hg for ﬁlter plate.

4. Calibrate the Bio-Plex system by following the prompts within the

Bio-Plex Manager

™

software. This can be done now or during an

assay incubation step.

5. Prepare 1x wash buffer. Mix 10x stock by inversion to ensure all salts are

in solution. Then dilute 1 part 10x wash buffer with 9 parts dH

Bio-Plex Pro

™

Assays

Quick Guide 6

Summary of content (4 pages)

PAGE 1
Bio-Plex Pro™ Assays Quick Guide 6 For use with Instruction Manual # Bio-Plex Pro Human Isotyping Assays 10028370 This guide can be used to prepare and run a full 1 x 96-well assay plate. For more information on a given step, refer to the complete instruction manual. New users can download the manual, which includes detailed instructions and a list of kit components, at www.bio-rad.com/bio-plex. IMPORTANT! Pay close attention to vortexing, shaking, and incubation instructions.
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Bio-Plex Pro Assay Quick Guide 6 6. Reconstitute the vial of standards in 781 µl of a diluent similar to your final sample type or matrix. Reconstitute the vial of quality controls in 250 µl of the same diluent, as shown below. Vortex for 5 sec and incubate all vials at once on ice for 30 min. Sample Type Diluent for Standards and Controls* Add BSA Serum and plasma Culture media, with serum Culture media, serum-free Isotyping diluent Culture media Culture media None None To 0.
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Bio-Plex Pro Assay Quick Guide 6 Running the Assay Note: Make sure all assay components are at RT before pipetting. 1. Prewet filter plate with 100 µl Bio-Plex assay buffer (skip for flat bottom). 2. Vortex the diluted (1x) beads. Add 50 µl to each well of the assay plate. 3. Wash the plate two times with 100 µl Bio-Plex wash buffer. 4. Vortex samples, standards, blank, and controls. Add 50 µl to each well. 5. Cover plate with sealing tape and protect from light with aluminum foil.
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Bio-Plex Pro Assay Quick Guide 6 14. Wash the plate three times with 100 µl wash buffer. 15. Resuspend beads in 125 µl assay buffer. Cover and shake at 850 ± 50 rpm for 30 sec. 16. Remove the sealing tape and read plate using the settings below. Instrument RP1 (PMT) DD Gates Bead Events Bio-Plex 100, 200* Low 5,000 (low), 25,000 (high) 50 Bio-Plex 3D* Standard Select MagPlex beads 50 Bio-Plex® MAGPIX™ N/A, use default instrument settings * A similar Luminex-based system may be used. 17.