Shreffler:Cisplatin Allergen: Difference between revisions

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# Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
# Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
# Remove pre-made stimulants (fMLP, Drug,  Anti-IgE) from freezer and let thaw.
# Remove pre-made stimulants (fMLP, Drug,  Anti-IgE) from freezer and let thaw.
# Place 5 mL RPMI in water bath for ~5-10 minutes.
# Place RPMI BSA aliquot in water bath for ~5-10 minutes.
# Prepare 15 5 mL PP tubes for conditions (A-O):
# Prepare 20 5 mL PS tubes for conditions (A-T):
#*A: Unstained
#*A: Unstained
#*B: RPMI
#*B: RPMI
Line 41: Line 41:
#*N: Cisplatin 4 (0.001X)
#*N: Cisplatin 4 (0.001X)
#*O: Cisplatin 5 (0.0001X)
#*O: Cisplatin 5 (0.0001X)
#*P: Oxyplatin 1 (1X)
#*Q: Oxyplatin 2 (0.1X)
#*R: Oxyplatin 3 (0.01X)
#*S: Oxyplatin 4 (0.001X)
#*T: Oxyplatin 5 (0.0001X)
#Prepare RPMI w/IL-3 as follows:
#Prepare RPMI w/IL-3 as follows:
#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
#*Add 8 μL of aliquot IL-3 (10 µL/mL) into 20 mL of warm RPMI. Mix well by inverting or vortex
# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
#* For "A: Unstained" and "B: RPMI," use RPMI (no IL-3)
#* For "C: IL-3," use RPMI IL-3 as is.
#* For "C: IL-3," use RPMI IL-3 as is.
#* For "D: fMLP," add 30 µL of 4 mM stock to 2370 µL RPMI IL-3 (1 part in 80), then add 20 µL of this dilution with 980 µL RPMI (1 part in 50) for final dilution.
#* For "D: fMLP," add 20 µL of stock to 980 µL RPMI IL-3.
#* For "E: Anti-IgE," add 30 µL of 200 µL stock to 2670 µL RPMI IL-3 (1 part in 90).  
#* For "E: Anti-IgE," add 10 µL of stock to 890 µL RPMI IL-3 (1 part in 90).  
#* For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.
#* For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.
#* For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
#* For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:
#* For "P: Oxyplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
# Gently mix blood by inverting green top tube 4 times.
#* For remainder of oxyplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
# '''IN THE HOOD''', add 250 μL of patient blood to all eleven tubes.
# Transfer 250 µL of each stimulant into separate PP tubes, labeled A-O
# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''
# Gently mix blood by inverting green top tube.
# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
# Transfer 250 μL of patient blood to each of the eleven PP tubes containing stimulants
#* To 900 uL staining buffer, add 90 μL each of:
# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).
#* CD63
# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:
#* CD203c
#: To 1240 µL staining buffer, add 190 μL each of:
#* CD123
#* CD63-FITC
#: and 12 μL of:
#* CD123-PC5
#* HLA-DR
#* HLA-DR-APC
#: '''Vortex'''
#: and 95 µL each of:
# After the 20 minutes of incubation, remove the samples.
#* CD203c-PC7
# Put samples on ice.
#* CRTH2-PE
# Add 110 μL of antibody cocktail to tubes B -> K '''(DO NOT ADD TO A!)'''.
#: Mix by vortex or inversion (if made in PP tube with cap)
# After the 20 minutes of incubation, remove the samples, putting them on ice.
# Add 100 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.
# Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
# Incubate for 20 min at 4°C. '''Protect from light'''.
# Incubate for 30 min at 4°C, taking care to limit exposure to light.
# Add 2 mL staining buffer.
# After incubation, wash samples with 2 mL staining buffer per tube.
# Invert tubes 3X to mix
# Invert tubes (covering opening w/ parafilm) 3X to mix
# Centrifuge for 5 minutes at 300g, RT
# Centrifuge for 5 minutes at 300g, RT
# Aspirate supernatant, taking care not to disturb cell pellet.
# 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'''.
# Add 2 mL of 1x FACS lysing solution one tube at a time, inverting until pellet is resuspended (cover opening w/ parafilm)
# Place tubes in the dark for 15 minutes or overnight in the refrigerator.
# Allow tubes to sit at RT (in dark) for 20 minutes. Following this, can suspend procedure by storing samples at 4ºC until ready to acquire flow data.
# Top off tube with staining buffer
# Add 1.5 mL staining buffer to each tube.
# Centrifuge for 5 minutes at 800g, RT.  
# Centrifuge for 5 minutes at 800g, RT.  
# Decant supernatant (do not double dump).
# Decant supernatant (do not double dump).
# Resuspend pellet in 50 μL staining buffer.
# Resuspend pellet in 75 μL staining buffer (final volume should be ~200 µL).
# Transfer samples to new PS tubes for flow acquisition.
# Aquire
# Aquire


Latest revision as of 09:43, 19 December 2011

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 our collaboration with Aleena Banerji in Allergy Associates and Oncology.

Materials

  • RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
  • IL-3 (R&D) 2 μg/mL (stock aliquots are 10 μg/mL)
  • 1 X FACS lysing solution (made from 10X stock with dH2O; 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
  • pipettes and tips
  • serological pipettes (5, 10, 25)
  • Platin drug (20X concentration)

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, Drug, Anti-IgE) from freezer and let thaw.
  3. Place RPMI BSA aliquot in water bath for ~5-10 minutes.
  4. Prepare 20 5 mL PS tubes for conditions (A-T):
    • A: Unstained
    • B: RPMI
    • C: IL-3
    • D: fMLP
    • E: Anti-IgE
    • F: Carboplatin 1 (1X)
    • G: Carboplatin 2 (0.1X)
    • H: Carboplatin 3 (0.01X)
    • I: Carboplatin 4 (0.001X)
    • J: Carboplatin 5 (0.0001X)
    • K: Cisplatin 1 (1X)
    • L: Cisplatin 2 (0.1X)
    • M: Cisplatin 3 (0.01X)
    • N: Cisplatin 4 (0.001X)
    • O: Cisplatin 5 (0.0001X)
    • P: Oxyplatin 1 (1X)
    • Q: Oxyplatin 2 (0.1X)
    • R: Oxyplatin 3 (0.01X)
    • S: Oxyplatin 4 (0.001X)
    • T: Oxyplatin 5 (0.0001X)
  5. Prepare RPMI w/IL-3 as follows:
    • Add 8 μL of aliquot IL-3 (10 µL/mL) into 20 mL of warm RPMI. Mix well by inverting or vortex
  6. Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
    • For "A: Unstained" and "B: RPMI," use RPMI (no IL-3)
    • For "C: IL-3," use RPMI IL-3 as is.
    • For "D: fMLP," add 20 µL of stock to 980 µL RPMI IL-3.
    • For "E: Anti-IgE," add 10 µL of stock to 890 µL RPMI IL-3 (1 part in 90).
    • For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.
    • For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
    • For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
    • For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
    • For "P: Oxyplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
    • For remainder of oxyplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
  7. Transfer 250 µL of each stimulant into separate PP tubes, labeled A-O
  8. Gently mix blood by inverting green top tube.
  9. Transfer 250 μL of patient blood to each of the eleven PP tubes containing stimulants
  10. Incubate tubes for 20 minutes in 37°C incubator (5% CO2).
  11. While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:
    To 1240 µL staining buffer, add 190 μL each of:
    • CD63-FITC
    • CD123-PC5
    • HLA-DR-APC
    and 95 µL each of:
    • CD203c-PC7
    • CRTH2-PE
    Mix by vortex or inversion (if made in PP tube with cap)
  12. After the 20 minutes of incubation, remove the samples, putting them on ice.
  13. Add 100 μL of antibody cocktail to tubes B -> K (DO NOT ADD TO A!).
  14. Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
  15. Incubate for 30 min at 4°C, taking care to limit exposure to light.
  16. After incubation, wash samples with 2 mL staining buffer per tube.
  17. Invert tubes (covering opening w/ parafilm) 3X to mix
  18. Centrifuge for 5 minutes at 300g, RT
  19. Aspirate supernatant, taking care not to disturb cell pellet.
  20. Add 2 mL of 1x FACS lysing solution one tube at a time, inverting until pellet is resuspended (cover opening w/ parafilm)
  21. Allow tubes to sit at RT (in dark) for 20 minutes. Following this, can suspend procedure by storing samples at 4ºC until ready to acquire flow data.
  22. Add 1.5 mL staining buffer to each tube.
  23. Centrifuge for 5 minutes at 800g, RT.
  24. Decant supernatant (do not double dump).
  25. Resuspend pellet in 75 μL staining buffer (final volume should be ~200 µL).
  26. Transfer samples to new PS tubes for flow acquisition.
  27. Aquire

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. Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation. Cell Res. 2005 May;15(5):325-35. DOI:10.1038/sj.cr.7290301 | PubMed ID:15916720 | HubMed [Hennersdorf]
  2. Knol EF, Mul FP, Jansen H, Calafat J, and Roos D. Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol. 1991 Sep;88(3 Pt 1):328-38. DOI:10.1016/0091-6749(91)90094-5 | PubMed ID:1716273 | HubMed [Knol]
  3. Shreffler WG. Evaluation of basophil activation in food allergy: present and future applications. Curr Opin Allergy Clin Immunol. 2006 Jun;6(3):226-33. DOI:10.1097/01.all.0000225165.83144.2f | PubMed ID:16670519 | HubMed [Shreffler]

All Medline abstracts: PubMed | HubMed

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