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 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

discuss this protocol

References

1. Redecha P, Franzke CW, Ruf W, Mackman N, and Girardi G. Neutrophil activation by the tissue factor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome. J Clin Invest. 2008 Oct;118(10):3453-61. DOI:10.1172/JCI36089 |

Contact

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