Shreffler:Basophil Activation

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 surface markers or even intracellular events (e.g. phosphorylation, oxidative burst, calcium flux) by flow cytometry. Separate markers (e.g. as CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation.

Materials

 * 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)

Stimulants

'''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

Peanut
 * 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)

Egg White
 * 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

Procedure
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
 * 1) Make Stimulants (BM, IL-3, and fMLP, anti-IgE) according to instructions above.
 * 2) Transfer 250 μL of warm RPMI to tube A.
 * 3) Transfer 250 μL of each stimulant as above to polypropylene tubes B-N.
 * 4) Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
 * 5) Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
 * 6) Prepare mAb staining cocktail.
 * 7) Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
 * 8) Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
 * 9) Incubate at 4°C for 30 minutes in the dark.
 * 10) Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300xg at 4°C.
 * 11) After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
 * 12) 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.
 * 13) Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
 * 14) After incubation, spin tubes at 800 x g for 10 minutes.  (If incubated overnight, consider washing with 2 mL staining buffer.)
 * 15) Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
 * 16) Store at 4°C until ready to acquire.

Discussion
discuss this protocol

Contact

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