title: Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry.
abstract: 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.
authors.affiliation: Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico. steinkamp@lanl.gov
authors.forename: J A
authors.initials: JA
authors.lastname: Steinkamp
authors.forename: N M
authors.initials: NM
authors.lastname: Lehnert
authors.forename: J F
authors.initials: JF
authors.lastname: Keij
authors.forename: B E
authors.initials: BE
authors.lastname: Lehnert
mesh_terms: D000954:Antigens, Surface / Q000032:analysis*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology; D005404:Fixatives; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005557:Formaldehyde; D005976:Glutaral; D006801:Humans; D008168:Lung / Q000166:cytology; D008863:Microspheres; D011108:Polymers
chemical_list: D000954:Antigens, Surface; D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D005557:Formaldehyde; D016650:Fluorescein-5-isothiocyanate; D005976:Glutaral; C003043:paraform