Shreffler:Basophil Sensitization

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(Materials)
(Procedure)
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==Procedure==
==Procedure==
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Attach appropriate labels (study ID & condition) to polypropylene tubes.
 
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  A. RPMI
 
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  B. IL-3 (BM)
 
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  C. FMLP
 
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  D. PN1
 
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  E. PN2
 
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  F. PN3
 
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  G. PN4
 
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  H. PN5
 
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  I. PN6
 
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  J. Anti-IgE
 
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  K. EW 1
 
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  L. EW 2
 
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  M. EW 3
 
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  N. EW 4
 
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# Make Stimulants (BM, IL-3, and fMLP, anti-IgE) according to instructions above.
 
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# Transfer 250 μL of warm RPMI to tube A.
 
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# Transfer 250 μL of each stimulant as above to polypropylene tubes B-N.
 
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# Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
 
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# Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
 
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# Prepare mAb staining cocktail.
 
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# Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
 
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# Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
 
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# Incubate at 4°C for 30 minutes in the dark.
 
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# Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300x''g'' at 4°C.
 
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# After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
 
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# Add 4 mL of 1X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O), dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
 
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# Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
 
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# After incubation, spin tubes at 800 x ''g'' for 10 minutes.  (If incubated overnight, consider washing with 2 mL staining buffer.)
 
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# Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
 
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# Store at 4°C until ready to acquire.
 
==Discussion==
==Discussion==

Revision as of 11:35, 28 May 2010

Contents

Overview

Materials

Procedure

Discussion

discuss this protocol

References

Error fetching PMID 15916720:
Error fetching PMID 1716273:
Error fetching PMID 16670519:
  1. Error fetching PMID 15916720: [Hennersdorf]
  2. Error fetching PMID 1716273: [Knol]
  3. Error fetching PMID 16670519: [Shreffler]
All Medline abstracts: PubMed HubMed


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