Following the preparation and dilution of the standards and mixing of the capture beads, transfer these reagents and test samples to the appropriate assay tubes for incubation and analysis.
1. Add 50 ¡L of the mixed capture beads to the appropriate assay tubes. Vortex the mixed capture beads before adding to the assay tubes.
2. Add 50 ¡L of the human Th1/Th2 PE-detection reagent to the assay tubes.
3. Add 50 ¡L of the human Th1/Th2 cytokine standard dilutions to the control assay tubes.
4. Add 50 ¡L of each test sample to the test assay tubes.
5. Incubate the assay tubes for 3 h at room temperature and protect from direct exposure to light.
6. Add 1 mL of wash buffer to each assay tube and centrifuge at 200g for 5 min.
7. Carefully aspirate and discard the supernatant from each assay tube.
8. Add 300 ¡L of wash buffer to each assay tube to resuspend the bead pellet.
9. Begin analyzing samples on a flow cytometer. Vortex each sample for 3-5 s immediately before analyzing on the flow cytometer.
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