With a few fluorophores, data are easy to interpret. With 30–40 fluorophores, analysis becomes challenging due to spillover and spectral overlap, requiring careful 𝘱𝘢𝘯𝘦𝘭 𝘥𝘦𝘴𝘪𝘨𝘯, 𝘤𝘰𝘮𝘱𝘦𝘯𝘴𝘢𝘵𝘪𝘰𝘯 or 𝘴𝘱𝘦𝘤𝘵𝘳𝘢𝘭 𝘶𝘯𝘮𝘪𝘹𝘪𝘯𝘨 [1]. 

𝗦𝗦𝗖, 𝗙𝗦𝗖, 𝗮𝗻𝗱 𝗟𝗶𝘃𝗲/𝗗𝗲𝗮𝗱 𝗦𝗲𝗽𝗮𝗿𝗮𝘁𝗶𝗼𝗻 
The initial gating step: forward scatter (FSC) and side scatter (SSC). 𝘍𝘚𝘊 reflects 𝘤𝘦𝘭𝘭 𝘴𝘪𝘻𝘦, while SSC correlates with internal complexity (e.g. 𝘨𝘳𝘢𝘯𝘶𝘭𝘢𝘳𝘪𝘵𝘺) [1]. Discrimination of cells from debris. Next, 𝘥𝘰𝘶𝘣𝘭𝘦𝘵𝘴 are removed using FSC-H versus FSC-A plots. A viability dye (e.g., 7AAD or fixable live/dead dyes) is then applied to 𝘦𝘹𝘤𝘭𝘶𝘥𝘦 𝘥𝘦𝘢𝘥 𝘤𝘦𝘭𝘭𝘴, which often bind antibodies nonspecifically [2]. 

𝗗𝗼𝘁 𝗣𝗹𝗼𝘁𝘀 𝗮𝗻𝗱 𝗛𝗶𝘀𝘁𝗼𝗴𝗿𝗮𝗺𝘀 
𝘋𝘰𝘵 𝘱𝘭𝘰𝘵𝘴: 𝘛𝘸𝘰-𝘥𝘪𝘮𝘦𝘯𝘴𝘪𝘰𝘯𝘢𝘭 (e.g., CD4 vs. CD8) plots used for visualizing correlations between markers and separating populations via quadrants. 
𝘏𝘪𝘴𝘵𝘰𝘨𝘳𝘢𝘮𝘴: 𝘖𝘯𝘦-𝘥𝘪𝘮𝘦𝘯𝘴𝘪𝘰𝘯𝘢𝘭 plots of fluorescence intensity (e.g., CD4 expression levels) are useful for comparing positive versus negative populations. 

𝗚𝗮𝘁𝗶𝗻𝗴 𝗦𝘁𝗿𝗮𝘁𝗲𝗴𝗶𝗲𝘀 𝗶𝗻 𝗛𝗶𝗴𝗵-𝗣𝗮𝗿𝗮𝗺𝗲𝘁𝗲𝗿 𝗣𝗮𝗻𝗲𝗹𝘀 
In high-parameter cytometry, hierarchical gating is critical: 
A. FSC/SSC to define cell population 
B. Singlet gating 
C. Viability gating 
D. Broad lineage marker gating (e.g., CD45) 
E. Subset or functional marker analysis (e.g., CD4, CD8) 

An example: Their gating strategy began with live single-cell selection (FSC-H vs FSC-A to exclude doublets), followed by viability gating, and then quantification of uPAR expression to identify senescent cells targeted by CAR T therapy [3]. Gating defines biological relevance in immunological studies.   

𝗦𝗼𝗳𝘁𝘄𝗮𝗿𝗲 𝗳𝗼𝗿 𝗚𝗮𝘁𝗶𝗻𝗴 𝗮𝗻𝗱 𝗩𝗶𝘀𝘂𝗮𝗹𝗶𝘇𝗮𝘁𝗶𝗼𝗻 
FlowJo: Widely used, manual gating 
FlowLogic: Flexible workflows and reporting 
Navios EX: Hardware-integrated software used in clinical cytometry 
Automated methods (e.g. GateNet) – a neural network trained to reproduce human gating. A future of reproducibility and scalability [4].   

𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻 
Do you prefer manual gating, or are you moving toward automated approaches? 

Stay tuned for 𝗗𝗮𝘆 𝟯𝟬: 𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗙𝗹𝗼𝘄 𝗖𝘆𝘁𝗼𝗺𝗲𝘁𝗿𝘆 

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀 
1. https://www.thermofisher.com/de/de/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-learning-center/flow-cytometry-resource-library/flow-cytometry-methods/spectral-flow-cytometry-fluorophore-selection.html 
2. https://www.bio-rad-antibodies.com/flow-cytometry-gating-strategies.html 
3. doi: 10.1038/s41586-020-2403-9 
4. DOI: 10.1016/j.compbiomed.2024.108820   

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