Attach appropriate labels (study ID & condition) to polypropylene tubes.
A. RPMI B. IL-3 (BM) C. FMLP D. PN1 E. PN2 F. PN3 G. PN4 H. PN5 I. PN6 J. Anti-IgE K. EW 1 L. EW 2 M. EW 3 N. EW 4
- Make Stimulants (BM, IL-3, and fMLP, anti-IgE) according to instructions above.
- Transfer 250 μL of warm RPMI to tube A.
- Transfer 250 μL of each stimulant as above to polypropylene tubes B-N.
- Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
- Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
- Prepare mAb staining cocktail.
- Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
- Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
- Incubate at 4°C for 30 minutes in the dark.
- Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300xg at 4°C.
- After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
- Add 4 mL of 1X FACS lysing solution (made from 10X stock with dH2O), dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
- Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
- After incubation, spin tubes at 800 x g for 10 minutes. (If incubated overnight, consider washing with 2 mL staining buffer.)
- Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
- Store at 4°C until ready to acquire.
- Hennersdorf F, Florian S, Jakob A, Baumgärtner K, Sonneck K, Nordheim A, Biedermann T, Valent P, and Bühring HJ. . pmid:15916720.
- Knol EF, Mul FP, Jansen H, Calafat J, and Roos D. . pmid:1716273.
- Shreffler WG. . pmid:16670519.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare