Flow cytometry is like an airport security checkpoint for cells: fluidics lines them up, lasers scan their ‘badges,’ and electronics log every detail instantly. This analogy captures how 𝘧𝘭𝘶𝘪𝘥𝘪𝘤𝘴, 𝘰𝘱𝘵𝘪𝘤𝘴, 𝘢𝘯𝘥 𝘦𝘭𝘦𝘤𝘵𝘳𝘰𝘯𝘪𝘤𝘴 work together to analyze individual cells per second. 

𝗣𝗿𝗶𝗻𝗰𝗶𝗽𝗹𝗲𝘀 
Cells are suspended in a fluid stream and pass individually through lasers. 𝘍𝘰𝘳𝘸𝘢𝘳𝘥 𝘴𝘤𝘢𝘵𝘵𝘦𝘳 𝘴𝘱𝘦𝘢𝘬𝘴 𝘰𝘧 𝘤𝘦𝘭𝘭 𝘴𝘪𝘻𝘦, 𝘴𝘪𝘥𝘦 𝘴𝘤𝘢𝘵𝘵𝘦𝘳 𝘮𝘦𝘢𝘴𝘶𝘳𝘦𝘴 𝘪𝘯𝘵𝘦𝘳𝘯𝘢𝘭 𝘤𝘰𝘮𝘱𝘭𝘦𝘹𝘪𝘵𝘺 (granularity), and fluorescence channels detect antigen-specific markers using 𝘧𝘭𝘶𝘰𝘳𝘰𝘱𝘩𝘰𝘳𝘦-𝘤𝘰𝘯𝘫𝘶𝘨𝘢𝘵𝘦𝘥 𝘢𝘯𝘵𝘪𝘣𝘰𝘥𝘪𝘦𝘴 [6]. 

𝗦𝘂𝗿𝗳𝗮𝗰𝗲 𝘃𝘀. 𝗜𝗻𝘁𝗿𝗮𝗰𝗲𝗹𝗹𝘂𝗹𝗮𝗿 𝗦𝘁𝗮𝗶𝗻𝗶𝗻𝗴 
Flow cytometry can detect 𝘴𝘶𝘳𝘧𝘢𝘤𝘦 𝘱𝘳𝘰𝘵𝘦𝘪𝘯𝘴 (e.g., CD4, CD16) as well as 𝘪𝘯𝘵𝘳𝘢𝘤𝘦𝘭𝘭𝘶𝘭𝘢𝘳 𝘱𝘳𝘰𝘵𝘦𝘪𝘯𝘴, 𝘴𝘶𝘤𝘩 𝘢𝘴 𝘤𝘺𝘵𝘰𝘬𝘪𝘯𝘦𝘴, after fixation and permeabilization. This allows researchers to link cell identity with functional state.  

𝗔𝗻𝗮𝗹𝘆𝘀𝗶𝘀 𝘃𝘀. 𝗦𝗼𝗿𝘁𝗶𝗻𝗴 
Cells can be analyzed to profile populations, or 𝘱𝘩𝘺𝘴𝘪𝘤𝘢𝘭𝘭𝘺 𝘴𝘦𝘱𝘢𝘳𝘢𝘵𝘦𝘥 𝘶𝘴𝘪𝘯𝘨 𝘧𝘭𝘶𝘰𝘳𝘦𝘴𝘤𝘦𝘯𝘤𝘦-𝘢𝘤𝘵𝘪𝘷𝘢𝘵𝘦𝘥 𝘤𝘦𝘭𝘭 𝘴𝘰𝘳𝘵𝘪𝘯𝘨 (𝘍𝘈𝘊𝘚) for downstream applications.  

𝗪𝗼𝗿𝗸𝗳𝗹𝗼𝘄 𝗮𝗻𝗱 𝗦𝗮𝗺𝗽𝗹𝗲 𝗣𝗿𝗲𝗽𝗮𝗿𝗮𝘁𝗶𝗼𝗻 
Workflow: cell isolation → counting → staining → cytometer acquisition → analysis/sorting. Dead cells are excluded with viability dyes. Panel design depends on the number of lasers, and spectral flow cytometry allows >30 parameters [6].  

𝗔𝗽𝗽𝗹𝗶𝗰𝗮𝘁𝗶𝗼𝗻𝘀 𝗕𝗲𝘆𝗼𝗻𝗱 𝗜𝗺𝗺𝘂𝗻𝗼𝗹𝗼𝗴𝘆 
Flow cytometry is applied outside immunology. Stüdenmann et al. 2022 tracked iPSC differentiation [3], while Bonser et al. (2021) purified airway Goblet cells [1].  

𝗔𝗽𝗽𝗹𝗶𝗰𝗮𝘁𝗶𝗼𝗻𝘀 𝗶𝗻 𝗜𝗺𝗺𝘂𝗻𝗼𝗹𝗼𝗴𝘆 
Kunath et al. (2025) used it to analyze B cell subsets after young vs. old HSC transplantation in mice [2]. Amor et al. 2024 quantified CD4+ and CD8+ T cells among CAR-T cells in mouse spleens under a high-fat diet [4]. Luu et al. (2018) measured IFN-γ+ cytotoxic T cells in response to butyrate exposure [5].  

Why do 𝘺𝘰𝘶 love flow cytometry: 
– designing high-dimensional panels 
– analyzing rare populations  
– seeing immune heterogeneity over time?   

Stay tuned for 𝗗𝗮𝘆 𝟮𝟳: 𝗙𝗹𝘂𝗼𝗿𝗼𝗽𝗵𝗼𝗿𝗲 𝗙𝘂𝗻𝗱𝗮𝗺𝗲𝗻𝘁𝗮𝗹𝘀, 𝗣𝗮𝗻𝗲𝗹 𝗗𝗲𝘀𝗶𝗴𝗻, 𝗮𝗻𝗱 𝗦𝗽𝗲𝗰𝘁𝗿𝗮𝗹 𝗙𝗹𝗼𝘄 𝗩𝗶𝘀𝘂𝗮𝗹𝗶𝘇𝗮𝘁𝗶𝗼𝗻   

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀 
1. doi: 10.1165/rcmb.2020-0149MA 
2. DOI: 10.1186/s12979-025-00507-x
3. DOI: 10.1161/CIRCULATIONAHA.122.060124 
4. DOI: 10.1038/s43587-023-00560-5 
5. DOI: 10.1038/s41598-018-32860-x 
6. doi: 10.1002/eji.201970107   

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