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 measur ements by flow cytometry is background autofluorescence. Methods: Using human lung fibroblasts (HLFs) as an autofluorescent cell mod el, unfixed HLFs and HLFs fixed in methanol, ethanol, formaldehyde, parafor maldehyde and glutaraldehyde were analyzed by phase-sensitive flow cytometr y to compare their fluorescence intensity and lifetime histograms. Based on these results, a surface antigen on HLFs was labeled with a fluorescein is othiocyanate (FITC) conjugated antibody and fixed in glutaraldehyde, and th e 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, wi th coefficients of variation 25-35%. Since the autofluorescence lifetime hi stograms of unfixed and fixed HLFs partially overlapped the 3.5 ns lifetime histogram of FITC-labeled microspheres, which were used to approximate FIT C-antibody labeling of HLFs, the ability to resolve FITC-labeled probe, bas ed on differences in the FITC and autofluorescence lifetimes, was severely Limited. When HLFs labeled with an FITC-antibody cell-surface marker were f ixed in glutaraldehyde (autofluorescence lifetime 0.9-1.4 ns, coefficient o f variation approximate to 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 meas urement resolution of surface antigens on highly autofluorescent, glutarald ehyde-fixed cells. Cytometry 37: 275-283, 1999. Published 1999 Wiley-Liss, Inc.dagger