Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.
Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies.
Cytonome, Inc., Boston, MA 02210, USA. rhulspas@cytonomest.com
Ruud
R
Hulspas
David
D
Dombkowski
Frederic
F
Preffer
Derick
D
Douglas
Brian
B
Kildew-Shah
John
J
Gilbert
D001710:Biotin / Q000737:chemistry; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D002469:Cell Separation; D004396:Coloring Agents / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D010799:Phycoerythrin / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D013601:T-Lymphocytes / Q000166:cytology*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology
D004396:Coloring Agents; D005456:Fluorescent Dyes; D011137:Polystyrenes; D010799:Phycoerythrin; D001710:Biotin