Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.
BACKGROUND
To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes.


METHODS
The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations.


RESULTS
With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed.


CONCLUSIONS
With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.
Department of Otorhinolaryngology/Plastic Surgery, University of Leipzig, Leipzig, Germany.
Andreas O H
AO
Gerstner
Dominik
D
Lenz
Wiebke
W
Laffers
Robert A
RA
Hoffman
Michael
M
Steinbrecher
Friedrich
F
Bootz
Attila
A
Tárnok
D052160:Benzothiazoles; D002232:Carbocyanines; D003116:Color; D005456:Fluorescent Dyes; D006801:Humans; D019044:Image Cytometry / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D016130:Immunophenotyping / Q000379:methods*; D007834:Lasers; D016131:Lymphocyte Subsets / Q000166:cytology*; D015203:Reproducibility of Results
D052160:Benzothiazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C011204:cyanine dye 7