Instrument Setup Data Acquisition and Analysis

FACSComp software is used for setting up the FACSCalibur flow cytometer daily. BD CellQuest software is required for analyzing samples and formatting data for subsequent analysis using the FCAP Array software. Setup for the FACSArray Bioanalyzer required only once a month. Sample acquisition is automated on the FACSCalibur, using the carousel loader, whereas the FACSArray instrument acquires the samples directly from the microplate wells. The data are analyzed using the FCAP Array Software. The outputs of the Flex Set assays are the standard curves for each assay and the tabulated results for each analytes. Fig. 6 shows the bead positions of a 9-plex cell signaling Flex Set combination. Fig. 7 demonstrates the kinetics of T cell activation with CD3/CD28 treatment using Jurkat cells.

4. Notes

1. The BD CBA is not recommended for use on stream-in-air instruments where signal intensities may be reduced, adversely affecting assay sensitivity. Stream-in-air instruments include the FACStar Plus and FACSVantage flow cytometers.

2. The antibody-conjugated beads will settle out of suspension over time. It is necessary to vortex the vial vigorously for 3-5 s before taking a bead suspension aliquot.

3. The human Th1/Th2 cytokine standards vials are stable until the kit expiration date. Following reconstitution, store the freshly reconstituted 10X bulk standard at 2-8°C and use within 12 h.

4. When running experiments with higher order multiplexes use the following instructions for reconstituting the soluble protein Flex Set standards. For multiplex experiments involving 10-20 soluble protein Flex Set assays, reconstitute each standard vial with 0.1 mL of assay diluent to prepare a 20X bulk standard. For multiplex experiments involving more than 20 soluble protein Flex Set assays, pour each standard protein sphere into a 15-mL conical tube and reconstitute all spheres together in 2 mL of assay diluent to prepare a top standard mixture.

5. To calibrate the flow cytometer and quantitate test samples, it is necessary to run the cytokine standards and the cytometer setup controls in each experiment.

6. For Flex Set assays that will be acquired on a FACSCalibur flow cytometer, it is recommended that additional dilutions of the standard be prepared (i.e., 1:512 and 1:1024) as it is possible that in multiplex experiments containing a large number of assays, the top standard and 1:2 standard dilution will not be analyzable by the FCAP Array software. In those cases, the top standard and 1:2 standard dilutions can be run on the experiment but will be excluded from the final analysis in the FCAP Array software.

7. Cell lysates may be stored at -70°C for up to 6 mo. Multiple freeze/thaw treatments of sample should be avoided.

8. It is necessary to analyze CBA samples on the day of the experiment. Prolonged storage of samples, once the assay is complete, can lead to increased background and reduced sensitivity.

9. The phospho-specific cell signaling Flex Set assays cannot be used in the same assay well with the total protein cell signaling Flex Set assays. An updated assay compatibility chart for the cell signaling Flex Sets is available at


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