Shreffler:Oxidative Burst

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 |SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma dissolved in DMSO at 5 mM (500X) and stored at -20°C.

Procedure

 * 1) Suspend PBMCs or other cells of interest at 1-10x106 cells/mL in appropriate medium.
 * 2) Make '2-10X' concentration (20-100 μM) of DHR in medium.
 * 3) Combine proportionately to cells in suspension (e.g. 5 mL PBMCs + 5 mL 2X DHR) in 15mL tube (final concentration 10 μM) and mix completely.
 * 4) Place in 37°C H2O bath for 5 minutes.
 * 5) Add stimulant of interest to cells at desired concentration.
 * 6) Incubate for 15-30 minutes.

Discussion
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Contact

 * Who has experience with this protocol?
 * Email Wayne Shreffler through OpenWetWare