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Published on: March 8, 2013
Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry
Andreas O H Gerstner1, Dominik Lenz, Wiebke Laffers
1Department of Otorhinolaryngology/Plastic Surgery, University of Leipzig, Leipzig, Germany.
Insights
This study adapted laser scanning cytometry (LSC) to measure cyanine 7 (Cy7) dyes, enabling enhanced multicolor immunophenotyping of leukocytes with improved discrimination of cell subsets.
Area of Science:
- Immunology
- Biotechnology
- Cell Biology
Background:
- Increased need for higher resolution in immunophenotyping to analyze immune system complexity.
- Laser scanning cytometry (LSC) offers slide-based multicolor visualization without cell sorting.
- Limitations in the number of measurable fluorochromes for leukocyte subset discrimination.
Purpose of the Study:
- To adapt the LSC for measuring cyanine 7 (Cy7) dyes.
- To increase the number of measurable fluorochromes for immunophenotyping.
- To enable advanced multicolor analysis of leukocyte subsets.
Main Methods:
- Modified LSC optical filters for photomultiplier tubes (PMT) 3 and 4 to accommodate allophycocyanin (APC) and Cy7.
- Tested Cy7 tandem dyes (phycoerythrin [PE]-Cy7, APC-Cy7) and other fluorochromes (FITC, PE, PE-Cy5, APC) with peripheral blood leukocytes.
- Developed and evaluated a six-color immunophenotyping panel.
Main Results:
- Successfully adapted LSC to measure Cy7 dyes with 488-nm or 633-nm laser excitation.
- Achieved FITC-level resolution for PE-Cy7, with slightly lower resolution for APC-Cy7.
- Observed prominent spillover for both tandem dyes into donor fluorochrome channels.
Conclusions:
- The adapted LSC system allows for the measurement of PE-Cy7 and APC-Cy7 tandem conjugates.
- This technical advancement facilitates six-color immunophenotyping using LSC.
- Enables more detailed analysis of leukocyte subsets with reduced sample volume.
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.
