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 oC in the dark)
• 1 X FACS Lysing Solution (made from 10X stock w/H2O, store at 4 oC, expires 1 week)
• PBS + 20 mM EDTA (sterile fliter, store at 4 oC, expires 1 week)
• Staining Buffer (PBS +2 mM EDTA + 0.5% BSA) (sterile filter, store at 4 oC, 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;store at 4 oC in the dark)
• Polypropylene tubes (BD Falcon cat #14959AA)
• Stimulant aliquots (at 20x concentration, distributed by Mt. Sinai; store frozen at -80 ºC)
• 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 oC ) 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 oC) 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 basophile 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:

   1. Prepare 10-fold dilutions of peanut extract stimulant (PE) (for conditions E-H below) as follows:
   2. Label three Eppendorf tubes as “PE 2-4”.
   3. Transfer 360 µL of basophile medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes.
   4. Transfer 40 µL from peanut extract tube 1 to tube 2 using 200 µL pipette and vortex for 5 seconds.
   5. 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).
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

5. Transfer 250 µL of warm (37 oC) RPMI to tubes A and B.
6. Transfer 250 µL of each warm (37 oC) 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 HLADR PE-Cy7) to a 5 mL polypropylene tube containing 900 µL staining buffer.
10. Add 50 µL cold (4º C) 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. Incubate at 4ºC for 30 min. in the dark.
13. Add 3 mL cold (4ºC) staining buffer to each tube.
14. Centrifuge for 5 min @ 300 x g at 4ºC (brake on).
15. After spin, carefully aspirate supernatant to within ~2mm of cells without disturbing pellet.
16. Add 4 mL of 1X FACS lysing solution to each tube with the cell pellet, mix thoroughly by inverting.
17. Incubate at room temperature in the dark for 15 minutes.
18. After incubation, spin tubes at 800 x g for 10 minutes.
19. Aspirate the supernatants carefully, then resuspend pellet in 200 µL of staining buffer and transfer to 1.5mL eppendorf tubes for shipment.
20. Attach a GlobalTrace Electronic Tracking System Study ID to each aliquot tube, and affix its matching label to the Laboratory Data Tracking Form.
21. Cover with parafilm.
22. Scan each GlobalTrace Electronic Tracking System label into the GlobalTrace Tracking System directly from the laboratory data form after samples have been stored. 24) Store at 4ºC and ship to Mt. Sinai as soon as possible. DO NOT FREEZE.

Discussion

discuss this protocol

References

1. Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. DOI:10.1016/0022-1759(94)90236-4 |

Contact

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