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Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent,
J A Steinkamp1, N M Lehnert, J F Keij
1Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico. steinkamp@lanl.gov
Insights
Phase-sensitive flow cytometry enhances immunofluorescence measurements by resolving surface antigens on autofluorescent cells. Glutaraldehyde fixation with this method effectively distinguishes labeled antigens from background autofluorescence.
Area of Science:
- Cellular and Molecular Biology
- Biophysical Techniques
- Immunology
Background:
- Background autofluorescence is a primary interference source in immunofluorescence measurements using flow cytometry.
- Accurate detection of surface antigens is crucial for various biological and medical applications.
Purpose of the Study:
- To evaluate the effectiveness of phase-sensitive flow cytometry in overcoming autofluorescence interference.
- To compare different fixation methods for human lung fibroblasts (HLFs) in the context of flow cytometry analysis.
- To determine if phase-resolved methods can improve the resolution of surface antigen labeling on highly autofluorescent cells.
Main Methods:
- Human lung fibroblasts (HLFs) were used as an autofluorescent cell model.
- Cells were analyzed unfixed or fixed with various agents (methanol, ethanol, formaldehyde, paraformaldehyde, glutaraldehyde).
- Phase-sensitive flow cytometry was employed to compare fluorescence intensity and lifetime histograms, alongside conventional methods for antibody-labeled cells.
Main Results:
- Autofluorescence lifetimes of unfixed and several fixed HLFs (ethanol, methanol, paraformaldehyde, formaldehyde) overlapped with fluorescein isothiocyanate (FITC)-labeled microspheres, limiting resolution.
- Glutaraldehyde fixation resulted in a distinct autofluorescence lifetime (0.9-1.4 ns) for HLFs.
- Phase-resolved analysis of glutaraldehyde-fixed, FITC-antibody-labeled HLFs successfully resolved the labeled antigen from background autofluorescence.
Conclusions:
- Phase-sensitive detection significantly improves the resolution of immunofluorescence measurements for surface antigens on highly autofluorescent cells.
- Glutaraldehyde fixation is a suitable method for preparing highly autofluorescent cells for analysis using phase-sensitive flow cytometry.
- This technique enhances the reliability of flow cytometry in identifying specific cellular markers in challenging biological samples.
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.
