The advent of modern flow cytometers and flow cytometric methods has accelerated researchers’ ability to enhance the resolution of samples analyzed. Within a span of 20 years, flow cytometry panels have ballooned from a handful of channels to well over 40.
The theoretical limit of flow panels now lies not in the available fluorophores or the laser lines to detect them but in our ability to prepare the sample with enough stringency to differentiate so many channels. The challenge now becomes how to prepare samples to enable multiparametic panels without sacrificing the overall resolution. Sahir et al. in Cytometry Part A exemplify how Laminar Wash technology directly improves the preparation of complex flow cytometric experiments. The authors demonstrate through a combination of Laminar Wash, advanced algorithms, and analysis software that they could resolve every major component of circulating peripheral blood mononuclear cells (PBMCs) using a 43 marker panel.
The panel was developed to directly analyze one million PBMCs isolated from a healthy donor, identifying over 130 unique cell types, including cells that comprise all major immune subsets and hematopoietic stem cells. Spectral analysis showed that the signals detected were due to discreet populations instead of non-specific binding. Additionally, the signals observed correlated well with fluorescence minus one controls, indicating that the signals correspond to expected populations in the sample. In order to test for the robustness of the panel, the stain was repeated seven times with different cell densities. For all replicates, identical populations were detected with similar frequencies within each population. This reproducibility indicates that the cell preparation protocol relying on Laminar Wash enables the panel to consistently and clearly identify all major PBMC components.
Successfully executing a 43 color panel is a technically demanding task, one that is compounded by confounding issues like non-specific binding and the presence of cell debris. Washing away these commingling curmudgeons while reducing hands-on steps is key to enabling such multifaceted studies. This study demonstrates that a panel comprised of a large number of antibody types can generate notably high reproducibility when Laminar Wash technology facilitates sample preparation, even when cells are present at low abundance.
Publication: Sahir F et al. December 18, 2020. “Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.” Cytometry A. Online ahead of print.