Flow cytometry analysis of human immune cells is a powerful tool for correctly diagnosing the root causes of certain diseases. This analysis relies on simultaneous detection of multiple fluorescent labels conjugated to antibodies specific for protein biomarkers expressed by the cell. The human immune system is sophisticated, requiring upwards of 30 parameters for a complete diagnosis of certain diseases such as leukemia, lymphoma, and COVID-19. Conventional flow cytometry is limited to approximately 20 parameters and is therefore not able to support analysis of more complex immune responses. To overcome this deficiency, spectral flow cytometers have been developed with the ability to analyze up to 40 cell parameters from a single sample. However, designing panels that maximize these analytical capabilities remains challenging due to the limited number of available fluorescent labels. To accommodate the enhanced multiplexing capabilities of the spectral flow cytometer, additional new dyes with unique spectral properties are needed.
In a recent study published by Cytek in collaboration with Biotium, Jiang et al. focused on the development of flow cytometry reagents using CF® Dyes, a new class of small organic dyes developed with improved solubility, brightness, and stability over currently available fluorophores. Initial studies validated the use of CD19-CF®633 and CD45-CF®405L as accurate biomarkers in a 4-color panel with CD3-FITC and CD4-PE-Cy®7 for immune cell phenotyping in whole blood samples. The authors then assessed the relative performance of CF® Dyes by comparing the stain index (SI) of CD4 antibody conjugated to 25 CF® Dyes with Alexa Fluor® 532 and Alexa Fluor®700 as control variables. Results show most of the CF® Dyes have higher SIs than Alexa Fluor® 532 and 11 CF® Dyes have higher SIs than Alexa Fluor® 700. Notably, CF®568, CF®633, and CF®640R showed exceptionally high SIs over 300 while Alexa Fluor® 532 and Alexa Fluor® 700 had SI values of 14 and 81, respectively. The authors conclude that CF® Dyes significantly expand the capability of spectral flow cytometry technology, paving the way for using high dimensional panels to study complex immune responses.
Learn more about our next generation CF® Dyes and how they take full advantage of spectral flow cytometry technology. Also learn more about our Mix-n-Stain CF® Dye Antibody Labeling Kits, and our wide-variety of CF® Dye succinimidyl esters and other reactives.
Jiang, J., Li, X., Mao, F., Wu, X., & Chen, Y. (2020). Small molecular fluorescence dyes for immuno cell analysis. Analytical Biochemistry, 114063. https://doi.org/10.1016/j.ab.2020.114063