Fluorochrome choices for multi-color flow cytometry
| Autor: | Tomas Kalina, Katerina Rejlova, Marta Martin-Ayuso, Alberto Orfao, Andrea Mayado, Juan Flores-Montero, Luzalba Sanoja-Flores, Vincent H.J. van der Velden, Paula Fernandez, Martin Perez-Andres, Sebastian Böttcher, Jaques J.M. van Dongen, Lukasz Sedek, Alba Corral-Mateos |
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| Přispěvatelé: | Immunology, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), European Commission |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Immunology Fluorochromes Flow cytometry Immunophenotyping Single tube 03 medical and health sciences 0302 clinical medicine EuroFlow medicine Immunology and Allergy Humans Fluorescent Dyes Allophycocyanin medicine.diagnostic_test Chemistry Antibody panel combination Multicolor flow cytometry Flow Cytometry Fluorescence Fluorescence intensity 030104 developmental biology Color flow Cytometry 030215 immunology Biomedical engineering |
| Zdroj: | Journal of Immunological Methods, 475:Unsp 112618. Elsevier Journal of Immunological Methods, 475. ELSEVIER Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0022-1759 |
| Popis: | Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design. This work was supported by CB16/12/00400 grant (CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER) and grant PI16/00787 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain. TK and KR were supported by project NPU LO1604 and EU-Prague project CZ.2.16/3.1.00/24505. |
| Databáze: | OpenAIRE |
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