Preparation of PEDetection Reagents

The PE-detection reagent provided with each cell signaling Flex Set is a 50X bulk (1 ¡L/test) and should be mixed with other cell signaling Flex Set PE-detection reagent and diluted to their optimal volume per test (50 ¡L/test) before adding the PE-detection reagents to a given tube or assay well.

1. Determine the number of cell signaling Flex Sets to be used in each tube or assay well in the experiment (size of the multiplex).

2. Determine the number of assay tubes or wells to be run in the experiment.

3. Transfer 1 ¡L/test of each cell signaling Flex Set PE-detection reagent to a conical tube labeled "mixed PE-detection reagent."

4. Add detection reagent diluent to the mixed PE-detection reagent tube to bring the final volume to 50 ¡L/test.

Example: if five cell signaling Flex sets are being multiplexed for a given 20 test experiment, 1 ¡L/test of each cell signaling Flex Set PE-detection reagent needs to be added to the mixed PE-detection reagent tube (1 ¡L/test x 20 tests = 20 ¡L total volume of each BD CBA cell signaling Flex Set PE-detection reagent) and then add detection reagent diluent to bring the final volume to 50 ¡L/test by determining the remaining volume to add (the final volume of mixed PE-detection reagent is 20 tests x 50 ¡L/test = 1000 ¡L). A total of 100 ¡L of PE-detection reagent was added to the mixed PE-detection reagent tube previously listed when 20 ¡L total volume of each cell signaling Flex Set PE-detection reagent was added from the five cell signaling Flex Sets. The amount of detection reagent diluent to add is 1000 ¡L total volume - 100 ¡L of PE-detection reagents = 900 ¡L.

5. Vortex mixed PE-detection reagent briefly. Mixed PE-detection reagent is now ready to be used in the experiment.

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