Shreffler:Eosinophil Isolation

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'''Granulocyte Isolation''':
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==Overview==
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# Under hood, spray down the buffy bag and scissors with 70% EtOH and wait until dry. Then cut open the buffy coat bag and pour blood into a 50 mL Falcon tube.
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Dihydrorhodamine 123 (DHR) is a non-fluorescent, membrane permeable molecule that can loaded into cells to measure oxidative burst. Upon oxidation, it is converted to [http://en.wikipedia.org/wiki/Rhodamine rhodamine], which can be measured by flow cytometry in the fluorescein channel ([http://omlc.ogi.edu/spectra/PhotochemCAD/html/rhodamine123.html spectra]).
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==Materials==
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*15 mL polypropylene tubes
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*Medium
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*DHR-1,2,3 [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=D1054|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma] dissolved in DMSO at 5 mM (500X) and stored at -20°C.
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==Procedure==
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# Under hood, spray down the buffy bags and scissors with 70% EtOH and wait until dry.
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# Cut open the buffy coat bag and pur blood into a 50 mL Falcon tube.
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==Discussion==
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[[Talk:{{PAGENAME}}|discuss this protocol]]
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==References==
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==Contact==
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*Who has experience with this protocol?
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*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]
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[[Catagory:Shreffler:Resources]]
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[[Category:Protocol]]
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[[Category:Immunology]]

Revision as of 13:26, 4 May 2011

Contents

Overview

Dihydrorhodamine 123 (DHR) is a non-fluorescent, membrane permeable molecule that can loaded into cells to measure oxidative burst. Upon oxidation, it is converted to rhodamine, which can be measured by flow cytometry in the fluorescein channel (spectra).

Materials

  • 15 mL polypropylene tubes
  • Medium
  • DHR-1,2,3 Sigma dissolved in DMSO at 5 mM (500X) and stored at -20°C.

Procedure

  1. Under hood, spray down the buffy bags and scissors with 70% EtOH and wait until dry.
  2. Cut open the buffy coat bag and pur blood into a 50 mL Falcon tube.

Discussion

discuss this protocol

References

Contact


Catagory:Shreffler:Resources

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