Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay

Abstract

Background: Neutrophil subpopulations with enhanced oxidative reactivity have been described in a number of clinical and in vitro settings. In the dichlorofluorescin (DCFH) oxidation assay, it is essential to maintain cellular viability and plasma membrane integrity through all stages of sample preparation. The process of erythrocyte lysing is crucial because a number of commercial lysing reagents can increase leukocyte membrane permeability.

Methods: We assessed viability [propidium iodide (PI) method], DCFH oxidation, and CD11b expression of resting or in vitro-stimulated neutrophils exposed to six different red cell lysing procedures.

Results: Formaldehyde-containing reagents (Optilyse B, FACS Lyse, and Erythrolyse) but not hypotonic shock or ammonium chloride (NH(4)Cl) solutions rendered 91.4--99.8% of resting neutrophils PI positive, with concomitant reductions in dichlorofluorescein (DCF) fluorescence, suggesting efflux of the fluorochrome. However, when stimulated with N-formyl-methionyl-leucyl-phenylalanine or Yersinia enterocolitica and then treated with FACS Lyse or Erythrolyse, up to 69.9% of neutrophils remained PI negative and exhibited enhanced DCF fluorescence. CD11b expression of PI-positive and -negative neutrophils was comparable, suggesting that they were activated equally.

Conclusions: FACS Lyse and Erythrolyse can modify neutrophil plasma membrane integrity, whereas hypotonic shock and NH(4)Cl solutions retain cellular viability and are lysing methods of choice in evaluation of neutrophil respiratory burst by DCFH oxidation assay.