Troubleshooting Flow Cytometry, Day 30

𝗡𝗼 𝗘𝘃𝗲𝗻𝘁𝘀 𝗗𝗲𝘁𝗲𝗰𝘁𝗲𝗱 – 𝗖𝘆𝘁𝗼𝗺𝗲𝘁𝗲𝗿 𝗥𝘂𝗻𝗻𝗶𝗻𝗴 
𝗣𝗼𝘀𝘀𝗶𝗯𝗹𝗲 𝗖𝗮𝘂𝘀𝗲𝘀: 𝘊𝘭𝘰𝘨𝘨𝘦𝘥 𝘚𝘢𝘮𝘱𝘭𝘦 𝘕𝘰𝘻𝘻𝘭𝘦 
𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗦𝘁𝗲𝗽𝘀:  
𝘗𝘳𝘪𝘮𝘦 𝘵𝘩𝘦 𝘚𝘺𝘴𝘵𝘦𝘮: Run sheath fluid through the system. 
𝘊𝘭𝘦𝘢𝘯 𝘵𝘩𝘦 𝘕𝘰𝘻𝘻𝘭𝘦: Use appropriate cleaning solutions, to clean the nozzle [1]. 
𝘊𝘩𝘦𝘤𝘬 𝘍𝘭𝘶𝘪𝘥 𝘓𝘦𝘷𝘦𝘭𝘴: Ensure that sheath and waste fluids are at adequate levels. 

𝗣𝗼𝗽𝘂𝗹𝗮𝘁𝗶𝗼𝗻𝘀 𝗔𝗽𝗽𝗲𝗮𝗿𝗶𝗻𝗴 𝗚𝗿𝗮𝘆 𝗶𝗻 𝗟𝗶𝘃𝗲/𝗗𝗲𝗮𝗱 𝗦𝘁𝗮𝗶𝗻𝗶𝗻𝗴 
𝗣𝗼𝘀𝘀𝗶𝗯𝗹𝗲 𝗖𝗮𝘂𝘀𝗲𝘀: Antibody Staining Issues: If a population appears gray in the live/dead gate, it may indicate that the antibody was not added. 
𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗦𝘁𝗲𝗽𝘀: 
𝘝𝘦𝘳𝘪𝘧𝘺 𝘈𝘯𝘵𝘪𝘣𝘰𝘥𝘺 𝘈𝘥𝘥𝘪𝘵𝘪𝘰𝘯: Ensure that the antibody was added at the correct concentration. 

𝗖𝗼𝗺𝗽𝗲𝗻𝘀𝗮𝘁𝗶𝗼𝗻 𝗖𝗼𝗻𝘁𝗿𝗼𝗹 𝗦𝗵𝗼𝘄𝗶𝗻𝗴 𝗧𝘄𝗼 𝗣𝗲𝗮𝗸 
𝗣𝗼𝘀𝘀𝗶𝗯𝗹𝗲 𝗖𝗮𝘂𝘀𝗲𝘀: 𝘚𝘱𝘦𝘤𝘵𝘳𝘢𝘭 𝘖𝘷𝘦𝘳𝘭𝘢𝘱: Two peaks can indicate spectral overlap between fluorochromes. 
𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗦𝘁𝗲𝗽𝘀: 
𝘜𝘴𝘦 𝘚𝘪𝘯𝘨𝘭𝘦-𝘚𝘵𝘢𝘪𝘯𝘦𝘥 𝘊𝘰𝘯𝘵𝘳𝘰𝘭𝘴: Always include single-stained controls [2].   

𝗗𝗼𝘂𝗯𝗹𝗲 𝗣𝗼𝘀𝗶𝘁𝗶𝘃𝗲 𝗣𝗼𝗽𝘂𝗹𝗮𝘁𝗶𝗼𝗻𝘀 𝗳𝗼𝗿 𝗖𝗗𝟰 𝗮𝗻𝗱 𝗖𝗗𝟴 
𝗣𝗼𝘀𝘀𝗶𝗯𝗹𝗲 𝗖𝗮𝘂𝘀𝗲𝘀: 𝘉𝘪𝘰𝘭𝘰𝘨𝘪𝘤𝘢𝘭 𝘗𝘩𝘦𝘯𝘰𝘮𝘦𝘯𝘰𝘯: Rarely, certain T cell subsets can express both CD4 and CD8 markers. 
𝗜𝗻𝘁𝗲𝗿𝗽𝗿𝗲𝘁𝗮𝘁𝗶𝗼𝗻: 
𝘊𝘰𝘯𝘧𝘪𝘳𝘮 𝘉𝘪𝘰𝘭𝘰𝘨𝘪𝘤𝘢𝘭 𝘙𝘦𝘭𝘦𝘷𝘢𝘯𝘤𝘦: Refer to literature to determine if the double-positive population is known in biology. Studies have documented [3]. 

𝗦𝗽𝗶𝗹𝗹𝗼𝘃𝗲𝗿 𝗕𝗲𝘁𝘄𝗲𝗲𝗻 𝗙𝗹𝘂𝗼𝗿𝗼𝗰𝗵𝗿𝗼𝗺𝗲𝘀 
𝗣𝗼𝘀𝘀𝗶𝗯𝗹𝗲 𝗖𝗮𝘂𝘀𝗲𝘀: 𝘚𝘱𝘦𝘤𝘵𝘳𝘢𝘭 𝘖𝘷𝘦𝘳𝘭𝘢𝘱: Spillover occurs when the emission spectra of different fluorochromes overlap. 
𝗧𝗿𝗼𝘂𝗯𝗹𝗲𝘀𝗵𝗼𝗼𝘁𝗶𝗻𝗴 𝗦𝘁𝗲𝗽𝘀: 
𝘖𝘱𝘵𝘪𝘮𝘪𝘻𝘦 𝘍𝘭𝘶𝘰𝘳𝘰𝘤𝘩𝘳𝘰𝘮𝘦 𝘚𝘦𝘭𝘦𝘤𝘵𝘪𝘰𝘯: Choose fluorochromes with minimal spectral overlap. 
𝘈𝘥𝘫𝘶𝘴𝘵 𝘊𝘰𝘮𝘱𝘦𝘯𝘴𝘢𝘵𝘪𝘰𝘯: Fine-tune compensation settings [4]. 

𝗜𝗺𝗽𝗼𝗿𝘁𝗮𝗻𝗰𝗲 𝗼𝗳 𝗖𝗼𝗻𝘁𝗿𝗼𝗹𝘀 𝗮𝗻𝗱 𝗕𝗲𝘀𝘁 𝗣𝗿𝗮𝗰𝘁𝗶𝗰𝗲𝘀 
In previous posts: the use of appropriate controls – Day 28, Day 29 – gating strategies, and Day 26 – the importance of fixation.  

𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻: 
Which flow cytometry issue do you encounter most frequently? 

Stay tuned for 𝗗𝗮𝘆 𝟯𝟭: 𝗖𝗲𝗹𝗹 𝗖𝘂𝗹𝘁𝘂𝗿𝗲 𝗘𝘀𝘀𝗲𝗻𝘁𝗶𝗮𝗹𝘀 – 𝗦𝘁𝗲𝗿𝗶𝗹𝗶𝘁𝘆 𝗮𝗻𝗱 𝗛𝗮𝗻𝗱𝗹𝗶𝗻𝗴 

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀 
1. https://bitesizebio.com/28983/troubleshooting-no-events-cytometer/ 
2. https://www.bio-rad-antibodies.com/flow-cytometry-comp-controls.html 
3. doi: 10.1038/s41598-021-03013-4 
4. https://fluorofinder.com/compensation-flow-cytometry/ 

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