Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry

BACKGROUND: The primary source of interference in immunofluorescence measurements by flow cytometry is background autofluorescence. METHODS: Using human lung fibroblasts (HLFs) as an autofluorescent cell model, unfixed HLFs and HLFs fixed in methanol, ethanol, formaldehyde, paraformaldehyde and glutaraldehyde were analyzed by phase-sensitive flow cytometry to compare their fluorescence intensity and lifetime histograms. Based on these results, a surface antigen on HLFs was labeled with a fluorescein isothiocyanate (FITC) conjugated antibody and fixed in glutaraldehyde, and the cells were analyzed by conventional and phase-resolved methods. RESULTS: The lifetimes of unfixed and ethanol-, methanol-, paraformaldehyde- and formaldehyde-fixed HLFs were in the 1.7-1.9 nanosecond (ns) range, with coefficients of variation 25-35%. Since the autofluorescence lifetime histograms of unfixed and fixed HLFs partially overlapped the 3.5 ns lifetime histogram of FITC-labeled microspheres, which were used to approximate FITC-antibody labeling of HLFs, the ability to resolve FITC-labeled probe, based on differences in the FITC and autofluorescence lifetimes, was severely limited. When HLFs labeled with an FITC-antibody cell-surface marker were fixed in glutaraldehyde (autofluorescence lifetime 0.9-1.4 ns, coefficient of variation approximately 11%) and analyzed by phase-resolved methods, the results showed that FITC-antibody labeling could be readily resolved from background autofluorescence. CONCLUSIONS: Phase-sensitive detection improves the immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells. Cytometry 37: 275-283, 1999. Published 1999 Wiley-Liss, Inc.