Shreffler:Basophil tSyk

Overview
Spleen tyrosine kinase (SYK) is a key signaling molecule downstream of many ITAM-containing receptors, including the β and γ chains of the FcεRI. Several investigators have reported that levels of SYK may correlate with basophil responsiveness and that SYK is degraded as a consequence of receptor-induced anergy.

Materials

 * Peripheral blood sample: 3.5 mL in Sodium Heparin (green top) collection tubes
 * RPMI medium (store at 4°C in the dark)
 * 1 X FACS Lysing Solution (made from 10X stock w/ dH2O, store at 4°C, expires 1 week)
 * 1 X PERM/WASH (Cat. No. 554723; made from 10X stock w/ dH2O, store at 4°C, expires 1 week)
 * PBS + 20 mM EDTA (sterile fliter, store at 4°C, expires 1 month)
 * Staining Buffer (PBS +2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C, aliquot in hood, expires 2 months)
 * Monoclonal antibodies (CD63-FITC, CD203c, CD123 PE-Cy5, HLA-DR PE-Cy7, CD69-APCCy7, CD3-, CD14-, CD19-, CD41a-APC, tSyk-FITC; store at 4°C in the dark)
 * MESF beads (Bangs Laboratories)
 * Polypropylene tubes (BD Falcon cat #14959AA)
 * Stimulant aliquots (at 20x concentration, distributed by Mt. Sinai; store frozen at -80ºC)
 * Basophil medium (RPMI w/ 40 ng/mL human IL-3) at 200 μL pre-made aliquots
 * Basophil medium w/20 μM fMLP at 40 μL pre-made aliquots
 * Basophil medium w/ 20 mg/mL anti-IgE at 40 mL pre-made aliquots
 * Basophil medium with stimulant:
 * For CoFAR3: Basophil medium w/ 20 μg/mL egg white antigen (egg white) at 40 μL pre-made aliquots
 * For CoFAR1 and 4: Basophil medium w/ 20 μg/mL peanut extract antigen (PE) at 40 μL premade aliquots]

Procedure

 * 1) Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and corresponding participant labels.
 * 2) Remove the 2 mL Cryotube with 200 μL of the 20x basophil media, thaw and add 1.8 mL of warm 37°C ) RPMI medium. Close the stopper and vortex it for 5 sec. This is the 2X basophil media to be used for incubation with the whole blood and for preparation of the egg or peanut allergen dilutions (see below).
 * 3) Remove stimulants from freezer, thaw, pulse-spin in microcentrifuge to ensure full volume recovery and add 360 μL warm (37°C) RPMI medium.
 * For CoFAR3: Prepare 10-fold dilutions of egg white stimulant (for conditions E-H below) as follows:
 * 1) Label three Eppendorf tubes as “egg white 2-4”.
 * 2) Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the egg white stimulant tubes.
 * 3) Transfer 40 μL from egg white tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds.
 * 4) Transfer 40 μL from tube 2 to tube 3 using 200 μL pipette and vortex for 5 seconds. Continue making 10-fold dilutions in the same manner until all four egg white dilutions have been prepared (tubes 1-4).
 * For CoFAR4: Prepare 10-fold dilutions of peanut extract stimulant (PE) (for conditions E-H below) as follows:
 * 1) Label three Eppendorf tubes as “PE 2-4”.
 * 2) Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes.
 * 3) Transfer 40 μL from peanut extract tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds.
 * 4) Transfer 40 μL from tube 2 to tube 3 using 200 μL pipette and vortex for 5 seconds. Continue making 10-fold dilutions in the same manner until all four peanut extract dilutions have been prepared (tubes 1-4).
 * For CoFAR1: Prepare 10-fold dilutions of peanut extract stimulant (PE) (for conditions E-G below) and 10-fold dilution of egg white stimulant (EW) (for condition H below) as follows:
 * 1) Label two Eppendorf tubes as “PE 2-3”.
 * 2) Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes 2 and 3.
 * 3) Transfer 40 μL from peanut extract tube 1 (the original peanut extract tube with 360 μL of RPMI-step 3) to tube 2 using 200 μL pipette and vortex for 5 seconds.
 * 4) Transfer 40 μL from tube 2 to tube 3 using 200 μL pipette and vortex for 5 seconds. Now all peanut extract dilutions are prepared (tubes PE 1-3).
 * 5) Label one Eppendorf tubes as “EW 2”.
 * 6) Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to the egg white stimulant tube "EW 2".
 * 7) Transfer 40 μL from egg white tube 1 (the original egg white extract tube with 360 μL of RPMI-step 3) to tube 2 labeled as "EW 2" using 200 μL pipette and vortex for 5 seconds.
 * 8) Label (participant ID and condition) the 5 mL polypropylene tubes A-I:


 * Tube Condition
 * A tSyk
 * B RPMI
 * C Basophil medium
 * D fMLP
 * E Antigen 1
 * F Antigen 2
 * G Antigen 3
 * H Antigen 4
 * I Anti-IgE
 * J FITC beads (to be added immediately prior to acquisition)


 * 1) Transfer 250 μL of warm (37°C) RPMI to tubes A and B.
 * 2) Transfer 250 μL of each warm (37°C) stimulant to the appropriate polypropylene tube (C-I) according to the table above.
 * 3) Transfer 250 μL of patient whole blood to each tube (A-I). (note: If blood has been sitting for awhile, gently rock tubes 2-3 times). The total tube volume in each tube should now equal 500 µL.
 * 4) Incubate for 30 minutes at 37ºC in an incubator (5% CO2). Do not agitate!
 * 5) While incubating, mix the Ab cocktail: add 90μL of each Ab (CD63-FITC, CD203c, CD123 PE-Cy5, CD69-APC-Cy7, CD3-, CD14-, CD19-, CD41a-APC and 45μL of HLA-DR PE-Cy7) to a 5 mL polypropylene tube containing 900 μL staining buffer.
 * 6) Add 50 μL cold (4ºC) PBS w/20mM EDTA to each tube to stop degranulation.
 * 7) Stain cells by adding 150 μL of the prepared Ab cocktail to tubes B-I. Do not add to tube A!
 * 8) Stain tube A cells by adding 100 μL staining buffer, 10 μL anti-CD123 PE-Cy5 and 5 μL anti-HLA-DR PE-Cy7.
 * 9) Incubate at 4ºC for 30 min. in the dark.
 * 10) Add 3 mL cold (4ºC) staining buffer to each tube.
 * 11) Centrifuge for 5 min @ 300 x g at 4ºC (brake on).
 * 12) After spin, carefully aspirate supernatant to within ~2mm of cells without disturbing pellet.
 * 13) Add 4 mL of 1X FACS lysing solution to each tube with the cell pellet, mix thoroughly by inverting.
 * 14) Incubate at room temperature in the dark for 15 minutes. Set tubes B-I aside.
 * 15) Working only with Tube A:
 * 16) Spin tube A at 800 x g for 10 minutes.
 * 17) Aspirate the supernatants carefully, then resuspend pellet with 500 μL 1 X Perm/Wash.
 * 18) Incubate at room temperature in the dark for 15 minutes.
 * 19) Spin tube A at 800 x g for 5 minutes.
 * 20) Aspirate the supernatants carefully, then resuspend pellet with 50 μL 1 X Perm/Wash.
 * 21) Add 1 μL anti-Syk FITC. Incubate at room temperature in dark for 15 minutes.
 * 22) Add 500 μL 1 X Perm/Wash..
 * 23) Spin all tubes at 800 x g for 10 minutes.
 * 24) Aspirate the supernatants carefully, then resuspend pellet with 75-100 μL of staining buffer and transfer to plate for acquisition.

Discussion
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Contact

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