Shreffler:SCIT Basophil Activation
Basophil activation in unfractionated samples such as 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. A feature of this protocol is to stimulate the basophils both in the presence and absence of autologous plasma.
This protocol is specifically for a mouse allergen SCIT study conducted at JHU.
- RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
- IL-3 (R&D) 2 μg/mL
- 1 X FACS lysing solution (made from 10X stock with dH2O; store at 4°C; expires in 1 month)
- PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 month)
- Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
- monoclonal antibodies (e.g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, HLA DR-PE-Cy7)
- stimulant aliquots (pre-made, 30 μL aliquots, distributed by Shreffler Lab; stored at -80°C)
- 5 mL round bottom polypropylene tubes (Falcon)
- 1.5 mL eppendorf tubes
- 15 and 50 mL conical tubes
- vacuum filter flasks (Corning #430186)
- pipettes and tips
- serological pipettes (5, 10, 25)
- Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours). Requires a minimum of 5 mL plus pipetting error (i.e., best to start with at least 5.5 mL).
- Remove pre-made stimulants (fMLP, Mouse allergen, Anti-IgE) from freezer.
- Label each stimulant and put aside to thaw.
- Centrifuge stimulants in the small centrifuge briefly.
- Aliquot 5 mL of RPMI into 15 mL conical tube
- Place RPMI in a 37°C water bath for a minimum of 10 minutes.
- Label eight eppendorf tubes for Mouse 2-5, and twenty 5 mL PP tubes for conditions (A1,2 - J1,2).
- Prepare Basophil medium (BM) as follows:
- Add 10 μL of IL-3 into 5 mL of warm RPMI (use 15 mL conical tube). Vortex
- Add 540 μL of warm BM to the following stimulants: anti-IgE and fMLP.
- Spin whole blood from the green tip heparin tube at 300 x g for 10 min. Transfer most of the plasma (supernatant) to 15 mL conical tube without disturbing cells and fill with PBS to wash. Invert.
- Spin the sample again for 10 min at 300 x g. Take off supernatant.
- While blood samples are spinning…
- Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
- Add 540 μL BM to Mouse 1 stimulant. Vortex
- Add 540 μL BM to each of the eppendorf tubes labeled Mouse 2-5.
- Transfer 60 μL from Mouse 1 stimulant into Mouse 2 eppendorf, vortex. Repeat this from Mouse 2 eppendorf into Mouse 3 eppendorf
- Continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 – Mouse 5). Vortex after each dilution.
- Transfer 250 μL of warm RPMI into tubes A1, A2 and B1, B2.
- Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (mouse allergen for tubes 'F1, F2'; basophil medium for tubes 'C1, C2', etc.).
- Gently mix whole blood cells by inverting green top tube 4 times.
- Divide evenly between two 15 mL conical tubes, '1' and '2'
- Add half of the retained plasma from above to tube '1' and a volume of RPMI equal to half of the plasma to tube '2'.
- Gently mix whole blood cells by inverting 4 times.
- Add 250 μL of patient blood '1' to the ten corresponding PP tubes (A1, B1, . . . J1).
- Add 250 μL of patient blood '2' to the ten corresponding PP tubes (A2, B2, . . . J2).
- Incubate tubes for 20 minutes in 37°C incubator (5% CO2). Do not shake tubes!
- While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
- To 1600 uL staining buffer, add 720 μL of premade antibody mix containing:
- CD63 - FITC (9 uL per test)
- CD203c - PE-Cy7 (9 uL per test)
- CD123 - PE-Cy5 (9 uL per test)
- HLA-DR - APC (4.5 uL per test)
- CRTH2 - PE (4.5 uL per test)
- After the 20 minutes of incubation, remove the samples.
- Immediately add 50 μL cold PBS-EDTA to each tube.
- Add 110 μL of antibody cocktail to tubes B -> K (DO NOT ADD TO A1 and A2!).
- Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
- Incubate for 30 min at 4°C. Protect from light.
- Add 2 mL staining buffer.
- Invert tubes 3X to mix
- Centrifuge for 5 minutes at 300g, RT
- Aspirate supernatant, taking care not to disturb cell pellet.
- Add 3 mL of 1x FACS lysing solution one tube at a time, recap and invert repeatedly until pellet is fully resuspended.
- Place tubes in the dark for 15 minutes or overnight in the refrigerator.
- Top off tube with staining buffer
- Centrifuge for 5 minutes at 800g, RT.
- Aspirate supernatant, taking care not to disturb pellet (leaving some residual supernatant is okay)
- Resuspend pellet in 50 μL staining buffer, making total liquid volume approximately 300 μL.
- Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
- Ship to Shreffler Lab at room temperature.
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- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare