Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry
✍ Scribed by J.A. Steinkamp; N.M. Lehnert; J.F. Keij; B.E. Lehnert
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 277 KB
- Volume
- 37
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
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 Ϸ11%) 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.