(New page: ==Overview== Basophil activation in unfractionated samples such as PBMC (note basophils are less dense the Ficoll) or whole blood can be measured by changes in the expression of cell surf...)
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Revision as of 11:34, 28 May 2010
- RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
- 1 X FACS lysing solution (made from 10X stock with dH2O; store at 4°C; expires in 1 week)
- PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
- Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
- desired monoclonal antibodies (e.g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, CD69-APC-Cy7, HLA DR-PE-CY7, CD3-APC, CD14-APC, CD19-APC, CD41a-APC)
May use other antigens for specific application. This will give an idea of the typical dose range we use for antigen-specific responses and controls.
Stimulant Preparation Basophil Medium (BM)= RPMI + 2 ng/mL human IL-3 (R&D, 203-IL-010/CF)
- Stock solution of IL-3 is 1,000X; (2 μg/mL aliquots stored in vials in -80°C); once vial is taken from -80°C, date, store 4°C and use within 1 month; vials stored in room 17-40
- For study, we need 2X stock (4 μM) in approximately 5 ml RPMI/subject
- 10 μL IL-3 in 5 mL RPMI
fMLP (Vendor, 47729-10MG-F) (Fisher, Nc9823482) – stored 4°C
- Stock solution at 4 mM in -30°C in 17-40
- We need 2X stock (2 μM) in 300 uL
- 1 μL of stock into 99 μL BM (100X) and vortex
- Take 15 μL of this intermediate dilution ad add to 285uL BM, vortex
Anti-IgE (Bethyl laboratories, A80-109A)
- Stock is 1 mg/mL (stored at 4°C in 17-40)
- We need 2 μg/mL (2X) in 300 μL
- 0.6 μL Anti-IgE in 300 uL BM
- Stock is 15 mg/mL
- We need out top dilution to be 107 pg/mL=10 μg/mL
- Prepare 6 dilutions starting at 20 μg/mL and serially diluting 10-fold.
- Add 0.5 uL peanute extract to 375 μL BM and vortex (Tube PN1)
- Proceed to make 5 serial dilutions with 270 μL BM in each tube, diluting with 30 uL from each previous tube (Tubes PN2-PN6)
- Stock is at 2 mg/mL
- Top dilution is 20 μg/mL
- Take 1μL EW stock and add to 99 μL BM and vortex
- Take 30 μL of this dilution and add to 270 μL BM (EW1)
- Serially dilute 10-fold 3 more times (EW 2-4) ie. 30μL of previous dilution in 270 μL BM
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