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.
4. 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)
   

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

Discussion

discuss this protocol

References

1. Macglashan D and Miura K. Loss of syk kinase during IgE-mediated stimulation of human basophils. J Allergy Clin Immunol. 2004 Dec;114(6):1317-24. DOI:10.1016/j.jaci.2004.08.037 |
2. MacGlashan DW Jr. Relationship between spleen tyrosine kinase and phosphatidylinositol 5' phosphatase expression and secretion from human basophils in the general population. J Allergy Clin Immunol. 2007 Mar;119(3):626-33. DOI:10.1016/j.jaci.2006.09.040 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare