Shreffler:JAX Basophil Activation

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(Procedure)
Current revision (13:27, 11 January 2011) (view source)
(Procedure)
 
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* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
-
* IL-3 (R&D) 2 μg/μL
+
* IL-3 (R&D) 2 μg/mL
-
* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)
+
* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 month)
-
* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)  
+
* 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)
* 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, CD69-APC-Cy7, HLA DR-PE-Cy7, CD3-APC, CD41a-APC)
+
* 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 -20°C)
+
* stimulant aliquots (pre-made, 30 μL aliquots, distributed by Shreffler Lab; stored at -80°C)
* 5 mL round bottom polypropylene tubes (Falcon)
* 5 mL round bottom polypropylene tubes (Falcon)
* 1.5 mL eppendorf tubes
* 1.5 mL eppendorf tubes
Line 25: Line 25:
==Procedure==
==Procedure==
-
# Obtain whole blood specimens collected in sodium heparin tube (green top).
+
# Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
-
# Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer. Label each stimulant and put aside to thaw.
+
# Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer.  
-
# Record the total volume of blood on the data form.
+
# Label each stimulant and put aside to thaw.
-
# Aliquot 5 mL of RPMI into 15 mL conical tube and place it in a 37°C water bath to keep warm (for the dilution of stimulants and for the Unstained Control).
+
# Centrifuge stimulants in the small centrifuge briefly.
-
# Label eppendorf tubes for stimulants and 5 mL PP tubes for conditions (A-K).
+
# Aliquot 5 mL of RPMI into 15 mL conical tube
-
# Prepare Basophil medium as follows:
+
# Place RPMI in a 37°C water bath for a minimum of 10 minutes.
-
#* Add 3 μL of IL-3 into 1.5 mL of warm RPMI (use 5 mL PP tube). '''Vortex'''
+
# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).
-
# Add 270 μL of warm RPMI to the following stimulants: anti-IgE, Mouse allergen, and fMLP stimulants. Pipette up and down to mix in cluster tube.
+
# Prepare Basophil medium (BM) as follows:
-
# Transfer 250 μL from each cluster tube to the corresponding eppendorf tube (labeled above -- mouse allergen for 'Mouse 1').
+
#* Add 5 μL of IL-3 into 2.5 mL of warm RPMI (use 5 mL PP tube). '''Vortex'''
-
# Add 135 μL of warm RPMI to each of the Calcium Ionophore and PMA tubes. Transfer both diluted stimulants into a single 1.5 mL eppendorf tube labeled CaI/PMA. '''Vortex'''
+
# Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP.  
 +
# Add 135 μL of warm BM to each of the Calcium Ionophore and PMA tubes.  
 +
# Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA. '''Vortex'''
# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
-
#* Transfer 270 μL of the Basophil medium prepared above to each of the Mouse 2-5 tubes.
+
#* Add 270 μL BM to Mouse 1 stimulant. '''Vortex'''
-
#* Transfer 30 μL from Mouse 1 stimulant into Mouse 2 tube, '''vortex'''.  Repeat this from Mouse 2 tube into Mouse 3 tube and 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.
+
#* Add 270 μL BM to each of the eppendorf tubes labeled Mouse 2-5.
-
#;[[Image:jax_stim.tiff]]
+
#* Transfer 30 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 A and B.
# Transfer 250 μL of warm RPMI into tubes A and B.
-
# Add 250 μL of each stimulant to the appropriate polypropylene tube (C-K) according to the table above.
+
# Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (mouse allergen for tube 'G'; basophil medium for tube 'C', etc.).
 +
#;[[Image:jax_stim.tiff]]
 +
# Gently mix blood by inverting green top tube 4 times.
# '''IN THE HOOD''', add 250 μL of patient blood to all eleven tubes.
# '''IN THE HOOD''', add 250 μL of patient blood to all eleven tubes.
# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).  '''Do not shake tubes!'''
# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).  '''Do not shake tubes!'''
# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
-
#* To 1.2 mL staining buffer, add 90 μL each of the CD63, CD203c, CD123, CD3 and CD41a and 45 μL each of the HLA-DR and CD69 mAbs. '''Vortex'''
+
#* To 800 uL staining buffer, add 360 μL of premade antibody mix sent by Shreffler Lab containing:
-
# After 20 minutes of incubation, remove the samples and add 50 μL cold PBS w/ 20 mM EDTA.
+
#* 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)
 +
#: '''Vortex'''
 +
# 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 A!)'''.
# Add 110 μL of antibody cocktail to tubes B -> K '''(DO NOT ADD TO A!)'''.
 +
# 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'''.
# Incubate for 30 min at 4°C. '''Protect from light'''.
-
# Quickly add 3-4 mL of 1x FACS lysing solution. Cover with parafilm and '''invert repeatedly until pellet is fully resuspended'''.
+
# 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.
# Place tubes in the dark for 15 minutes or overnight in the refrigerator.
-
# Centrifuge for 5 minutes at 800 x g at room temperature. Decant supernatant and resuspend pellet in 100 μL staining buffer.
+
# 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.
# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
# Ship to Shreffler Lab at room temperature.
# Ship to Shreffler Lab at room temperature.
 +
**Note that IL-3 stock at JAX is different concentration than at Shrefflab
==Discussion==
==Discussion==

Current revision

Contents

Overview

Basophil activation in unfractionated samples such as PBMC (note basophils are less dense than Ficoll) or 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.

This protocol is specifically for the JAX study.

Materials

  • 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)
  • parafilm
  • foil
  • pipettes and tips
  • serological pipettes (5, 10, 25)

Procedure

  1. Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
  2. Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer.
  3. Label each stimulant and put aside to thaw.
  4. Centrifuge stimulants in the small centrifuge briefly.
  5. Aliquot 5 mL of RPMI into 15 mL conical tube
  6. Place RPMI in a 37°C water bath for a minimum of 10 minutes.
  7. Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).
  8. Prepare Basophil medium (BM) as follows:
    • Add 5 μL of IL-3 into 2.5 mL of warm RPMI (use 5 mL PP tube). Vortex
  9. Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP.
  10. Add 135 μL of warm BM to each of the Calcium Ionophore and PMA tubes.
  11. Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA. Vortex
  12. Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
    • Add 270 μL BM to Mouse 1 stimulant. Vortex
    • Add 270 μL BM to each of the eppendorf tubes labeled Mouse 2-5.
    • Transfer 30 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.
  13. Transfer 250 μL of warm RPMI into tubes A and B.
  14. Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (mouse allergen for tube 'G'; basophil medium for tube 'C', etc.).
    Image:jax_stim.tiff
  15. Gently mix blood by inverting green top tube 4 times.
  16. IN THE HOOD, add 250 μL of patient blood to all eleven tubes.
  17. Incubate tubes for 20 minutes in 37°C incubator (5% CO2). Do not shake tubes!
  18. While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
    • To 800 uL staining buffer, add 360 μL of premade antibody mix sent by Shreffler Lab 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)
    Vortex
  19. After the 20 minutes of incubation, remove the samples.
  20. Immediately add 50 μL cold PBS-EDTA to each tube.
  21. Add 110 μL of antibody cocktail to tubes B -> K (DO NOT ADD TO A!).
  22. Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
  23. Incubate for 30 min at 4°C. Protect from light.
  24. Add 2 mL staining buffer.
  25. Invert tubes 3X to mix
  26. Centrifuge for 5 minutes at 300g, RT
  27. Aspirate supernatant, taking care not to disturb cell pellet.
  28. Add 3 mL of 1x FACS lysing solution one tube at a time, recap and invert repeatedly until pellet is fully resuspended.
  29. Place tubes in the dark for 15 minutes or overnight in the refrigerator.
  30. Top off tube with staining buffer
  31. Centrifuge for 5 minutes at 800g, RT.
  32. Aspirate supernatant, taking care not to disturb pellet (leaving some residual supernatant is okay)
  33. Resuspend pellet in 50 μL staining buffer, making total liquid volume approximately 300 μL.
  34. Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
  35. Ship to Shreffler Lab at room temperature.
    • Note that IL-3 stock at JAX is different concentration than at Shrefflab

Discussion

discuss this protocol

References

  1. Hennersdorf F, Florian S, Jakob A, Baumgärtner K, Sonneck K, Nordheim A, Biedermann T, Valent P, and Bühring HJ. . pmid:15916720. PubMed HubMed [Hennersdorf]
  2. Knol EF, Mul FP, Jansen H, Calafat J, and Roos D. . pmid:1716273. PubMed HubMed [Knol]
  3. Shreffler WG. . pmid:16670519. PubMed HubMed [Shreffler]
All Medline abstracts: PubMed HubMed


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