Shreffler:Basophil Sensitization

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(Overview)
(Materials)
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==Materials==
==Materials==
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* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
 
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* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)
 
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* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
 
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* Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
 
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* desired monoclonal antibodies (e.g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, CD69-APC-Cy7, HLA DR-PE-CY7, CD3-APC, CD14-APC, CD19-APC, CD41a-APC)
 
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Stimulants
 
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'''May use other antigens for specific application. This will give an idea of the typical dose range we use for antigen-specific responses and controls.'''
 
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Stimulant Preparation
 
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Basophil Medium (BM)= RPMI + 2 ng/mL human IL-3 (R&D, 203-IL-010/CF)
 
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* Stock solution of IL-3 is 1,000X; (2 μg/mL aliquots stored in vials in -80°C); once vial is taken from -80°C, date, store 4°C and use within 1 month; vials stored in room 17-40
 
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* For study, we need 2X stock (4 μM) in approximately 5 ml RPMI/subject
 
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** 10 μL IL-3 in 5 mL RPMI
 
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fMLP (Vendor, 47729-10MG-F) (Fisher, Nc9823482) – stored 4°C
 
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* Stock solution at 4 mM in -30°C in 17-40
 
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* We need 2X stock (2 μM) in 300 uL
 
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** 1 μL of stock into 99 μL BM (100X) and vortex
 
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* Take 15 μL of this intermediate dilution ad add to 285uL BM, vortex
 
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Anti-IgE (Bethyl laboratories, A80-109A)
 
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* Stock is 1 mg/mL (stored at 4°C in 17-40)
 
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* We need 2 μg/mL (2X) in 300 μL
 
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** 0.6 μL Anti-IgE in 300 uL BM
 
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Peanut
 
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* Stock is 15 mg/mL
 
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* We need out top dilution to be 10<sup>7</sup> pg/mL=10 μg/mL
 
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* Prepare 6 dilutions starting at 20 μg/mL and serially diluting 10-fold.
 
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** Add 0.5 uL peanute extract to 375 μL BM and vortex (Tube PN1)
 
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** Proceed to make 5 serial dilutions with 270 μL BM in each tube, diluting with 30 uL from each previous tube (Tubes PN2-PN6)
 
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Egg White
 
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* Stock is at 2 mg/mL
 
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* Top dilution is 20 μg/mL
 
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* Take 1μL EW stock and add to 99 μL BM and vortex
 
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* Take 30 μL of this dilution and add to 270 μL BM (EW1)
 
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* Serially dilute 10-fold 3 more times (EW 2-4) ie. 30μL of previous dilution in 270 μL BM
 
==Procedure==
==Procedure==

Revision as of 10:34, 28 May 2010

Contents

Overview

Materials

Procedure

Attach appropriate labels (study ID & condition) to polypropylene tubes.

 A. RPMI
 B. IL-3 (BM)
 C. FMLP
 D. PN1
 E. PN2
 F. PN3
 G. PN4
 H. PN5
 I. PN6
 J. Anti-IgE
 K. EW 1
 L. EW 2
 M. EW 3
 N. EW 4

  1. Make Stimulants (BM, IL-3, and fMLP, anti-IgE) according to instructions above.
  2. Transfer 250 μL of warm RPMI to tube A.
  3. Transfer 250 μL of each stimulant as above to polypropylene tubes B-N.
  4. Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
  5. Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
  6. Prepare mAb staining cocktail.
  7. Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
  8. Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
  9. Incubate at 4°C for 30 minutes in the dark.
  10. Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300xg at 4°C.
  11. After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
  12. Add 4 mL of 1X FACS lysing solution (made from 10X stock with dH2O), dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
  13. Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
  14. After incubation, spin tubes at 800 x g for 10 minutes. (If incubated overnight, consider washing with 2 mL staining buffer.)
  15. Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
  16. Store at 4°C until ready to acquire.

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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