Assay Procedure

Transfer the standards, capture beads, test samples, and PE-detection reagent to the appropriate assay tubes or wells for incubation and analysis. Flex Set standards are run in each experiment to allow quantitation of test samples.

1. Add 50 ¡L of the mixed capture beads to the appropriate assay tubes or wells. Vortex the mixed capture beads before adding them to the assay tubes or wells.

2. Add 50 ¡L of the mixed PE-detection reagent to the assay tubes or wells.

3. Add 50 ¡L of the cell signaling Flex Set standard dilutions to the control assay tubes or wells.

4. Add 50 ¡L of each denatured cell lysate test sample to the test assay tubes or wells.

5. For assays performed in tubes, mix assay tubes gently and incubate for 4 h at room temperature and protect from direct exposure to light. For assays performed in filter plate wells, mix the microwell plate for 15 min using a digital shaker at 500g and incubate plate for 4 h at room temperature and protect from direct exposure to light.

6. For assays run in tubes, add 1.0 mL of wash buffer to each assay tube and centrifuge at 200g for 5 min. For assays run in filter plate wells, apply the plate to the vacuum manifold and vacuum aspirate (do not exceed 10" Hg of vacuum) until wells are drained (2-10 s).

7. For assays run in tubes, carefully aspirate and discard the supernatant from each assay tube. For assays run in filter plate wells, proceed to step 8.

8. Add 300 ¡L of wash buffer to each assay tube or 150 ¡L of wash buffer to each assay well. Vortex assay tubes briefly or shake microwell plate on a digital shaker at 50g for 5 min to resuspend beads.

9. Begin analyzing samples on a flow cytometer. For assays run in tubes, it is recommended that each tube be vortexed briefly before analyzing on the flow cytometer.

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