Shreffler:Basophil tSyk: Difference between revisions
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*Staining Buffer (PBS +2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C, aliquot in hood, expires 2 months) | *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) | *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) | *Polypropylene tubes (BD Falcon cat #14959AA) | ||
*Stimulant aliquots (at 20x concentration, distributed by Mt. Sinai; store frozen at -80ºC) | *Stimulant aliquots (at 20x concentration, distributed by Mt. Sinai; store frozen at -80ºC) | ||
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#: '''For CoFAR3: Prepare 10-fold dilutions of egg white stimulant (for conditions E-H below) as follows:''' | #: '''For CoFAR3: Prepare 10-fold dilutions of egg white stimulant (for conditions E-H below) as follows:''' | ||
## Label three Eppendorf tubes as “egg white 2-4”. | ## Label three Eppendorf tubes as “egg white 2-4”. | ||
## Transfer 360 μL of | ## Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the egg white stimulant tubes. | ||
## Transfer 40 μL from egg white tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds. | ## Transfer 40 μL from egg white tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds. | ||
##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). | ##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 | #: '''For CoFAR4: Prepare 10-fold dilutions of peanut extract stimulant (PE) (for conditions E-H below) as follows:''' | ||
## Label three Eppendorf tubes as “PE 2-4”. | ## Label three Eppendorf tubes as “PE 2-4”. | ||
## Transfer 360 μL of | ## Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes. | ||
## Transfer 40 μL from peanut extract tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds. | ## Transfer 40 μL from peanut extract tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds. | ||
## 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). | ## 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). | ||
# Label (participant ID and condition) the 5 mL polypropylene tubes A-I: | #: '''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:''' | ||
## Label two Eppendorf tubes as “PE 2-3”. | |||
## Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes 2 and 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. | |||
## 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). | |||
## Label one Eppendorf tubes as “EW 2”. | |||
## Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to the egg white stimulant tube "EW 2". | |||
## 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. | |||
# Label (participant ID and condition) the 5 mL polypropylene tubes A-I:<br /> | |||
#:'''Tube Condition''' | #:'''Tube Condition''' | ||
#:A tSyk | #:A tSyk | ||
Line 47: | Line 56: | ||
#:H Antigen 4 | #:H Antigen 4 | ||
#:I Anti-IgE | #:I Anti-IgE | ||
#:J FITC beads | #:J FITC beads (to be added immediately prior to acquisition)<br /> | ||
# Transfer 250 μL of warm (37°C) RPMI to tubes A and B. | # Transfer 250 μL of warm (37°C) RPMI to tubes A and B. | ||
# Transfer 250 μL of each warm (37°C) stimulant to the appropriate polypropylene tube (C-I) according to the table above. | # Transfer 250 μL of each warm (37°C) stimulant to the appropriate polypropylene tube (C-I) according to the table above. |
Latest revision as of 13:02, 17 September 2009
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
- Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and corresponding participant labels.
- 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).
- 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:
- Label three Eppendorf tubes as “egg white 2-4”.
- Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the egg white stimulant tubes.
- Transfer 40 μL from egg white tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds.
- 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:
- Label three Eppendorf tubes as “PE 2-4”.
- Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes.
- Transfer 40 μL from peanut extract tube 1 to tube 2 using 200 μL pipette and vortex for 5 seconds.
- 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:
- Label two Eppendorf tubes as “PE 2-3”.
- Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to each of the peanut extract stimulant tubes 2 and 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.
- 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).
- Label one Eppendorf tubes as “EW 2”.
- Transfer 360 μL of basophil medium from the aliquots prepared in step 2 to the egg white stimulant tube "EW 2".
- 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.
- 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)
- Transfer 250 μL of warm (37°C) RPMI to tubes A and B.
- Transfer 250 μL of each warm (37°C) stimulant to the appropriate polypropylene tube (C-I) according to the table above.
- 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.
- Incubate for 30 minutes at 37ºC in an incubator (5% CO2). Do not agitate!
- 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.
- Add 50 μL cold (4ºC) PBS w/20mM EDTA to each tube to stop degranulation.
- Stain cells by adding 150 μL of the prepared Ab cocktail to tubes B-I. Do not add to tube A!
- Stain tube A cells by adding 100 μL staining buffer, 10 μL anti-CD123 PE-Cy5 and 5 μL anti-HLA-DR PE-Cy7.
- Incubate at 4ºC for 30 min. in the dark.
- Add 3 mL cold (4ºC) staining buffer to each tube.
- Centrifuge for 5 min @ 300 x g at 4ºC (brake on).
- After spin, carefully aspirate supernatant to within ~2mm of cells without disturbing pellet.
- Add 4 mL of 1X FACS lysing solution to each tube with the cell pellet, mix thoroughly by inverting.
- Incubate at room temperature in the dark for 15 minutes. Set tubes B-I aside.
- Working only with Tube A:
- Spin tube A at 800 x g for 10 minutes.
- Aspirate the supernatants carefully, then resuspend pellet with 500 μL 1 X Perm/Wash.
- Incubate at room temperature in the dark for 15 minutes.
- Spin tube A at 800 x g for 5 minutes.
- Aspirate the supernatants carefully, then resuspend pellet with 50 μL 1 X Perm/Wash.
- Add 1 μL anti-Syk FITC. Incubate at room temperature in dark for 15 minutes.
- Add 500 μL 1 X Perm/Wash..
- Spin all tubes at 800 x g for 10 minutes.
- Aspirate the supernatants carefully, then resuspend pellet with 75-100 μL of staining buffer and transfer to plate for acquisition.
Discussion
References
- 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 |
- 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