𝗙𝗹𝘂𝗼𝗿𝗼𝗽𝗵𝗼𝗿𝗲 𝗦𝗲𝗹𝗲𝗰𝘁𝗶𝗼𝗻 
Selecting fluorophores is crucial. For 𝘭𝘰𝘸-𝘦𝘹𝘱𝘳𝘦𝘴𝘴𝘦𝘥 𝘮𝘢𝘳𝘬𝘦𝘳𝘴, such as CD14 [1], it’s essential to 𝘤𝘩𝘰𝘰𝘴𝘦 𝘣𝘳𝘪𝘨𝘩𝘵𝘦𝘳 𝘧𝘭𝘶𝘰𝘳𝘰𝘱𝘩𝘰𝘳𝘦𝘴 to enhance signal detection. For highly expressed markers, selecting dimmer fluorophores can 𝘱𝘳𝘦𝘷𝘦𝘯𝘵 𝘴𝘪𝘨𝘯𝘢𝘭 𝘴𝘢𝘵𝘶𝘳𝘢𝘵𝘪𝘰𝘯 and ensure accurate quantification.  When using viability dyes like SirTubulin, it’s vital to consider their emission spectra to 𝘢𝘷𝘰𝘪𝘥 𝘰𝘷𝘦𝘳𝘭𝘢𝘱 𝘸𝘪𝘵𝘩 𝘰𝘵𝘩𝘦𝘳 𝘧𝘭𝘶𝘰𝘳𝘰𝘱𝘩𝘰𝘳𝘦𝘴 in the panel.  
Overlapping emission spectra can lead to compensation issues, making data interpretation challenging. 

𝗣𝗮𝗻𝗲𝗹 𝗗𝗲𝘀𝗶𝗴𝗻 
1. 𝘉𝘪𝘰𝘭𝘰𝘨𝘪𝘤𝘢𝘭 𝘘𝘶𝘦𝘴𝘵𝘪𝘰𝘯: Identify the cellular markers relevant to your research question. 
2. 𝘐𝘯𝘴𝘵𝘳𝘶𝘮𝘦𝘯𝘵 𝘊𝘰𝘯𝘧𝘪𝘨𝘶𝘳𝘢𝘵𝘪𝘰𝘯: Understand the lasers and filters of your cytometer to select compatible fluorophores. 
3. 𝘍𝘭𝘶𝘰𝘳𝘰𝘱𝘩𝘰𝘳𝘦 𝘚𝘦𝘭𝘦𝘤𝘵𝘪𝘰𝘯: Choose fluorophores with distinct emission spectra to minimize spectral overlap. 
4. 𝘈𝘯𝘵𝘪𝘣𝘰𝘥𝘺 𝘖𝘱𝘵𝘪𝘮𝘪𝘻𝘢𝘵𝘪𝘰𝘯: Titrate antibodies to determine optimal concentrations and specific binding. 
5. 𝘝𝘢𝘭𝘪𝘥𝘢𝘵𝘪𝘰𝘯: Test the panel on control samples to assess performance. 

𝗦𝗽𝗲𝗰𝘁𝗿𝗮𝗹 𝗙𝗹𝗼𝘄 𝗩𝗶𝘀𝘂𝗮𝗹𝗶𝘇𝗮𝘁𝗶𝗼𝗻 
Spectral flow cytometry allows analyzing more parameters simultaneously. Thermo Fisher Scientific’s SpectraViewer [3] or PTG Lab’s [4] enable researchers to visualize and compare the spectra of multiple fluorophores, selecting compatible dyes and optimizing panel design. 

𝗔𝗽𝗽𝗹𝗶𝗰𝗮𝘁𝗶𝗼𝗻 𝗘𝘅𝗮𝗺𝗽𝗹𝗲: 𝗦𝗶𝗿𝗧𝘂𝗯𝘂𝗹𝗶𝗻 𝗶𝗻 𝗔𝗶𝗿𝘄𝗮𝘆 𝗘𝗽𝗶𝘁𝗵𝗲𝗹𝗶𝗮𝗹 𝗖𝗲𝗹𝗹𝘀 
In a study [2], antibody panels were developed to characterize and isolate 4 major airway epithelial subsets, including ciliated cells, from human bronchial epithelial cell cultures. 

𝗣𝗲𝗿𝘀𝗼𝗻𝗮𝗹 𝗶𝗻𝘁𝗲𝗿𝗲𝘀𝘁: 𝗜𝗺𝗺𝘂𝗻𝗼𝘀𝗲𝗻𝗲𝘀𝗰𝗲𝗻𝗰𝗲 𝗠𝗮𝗿𝗸𝗲𝗿𝘀 
Currently, I am curious about immunosenescence markers for a panel aimed at understanding aging immune system and potential therapeutic targets.   

𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻 
What tools do 𝘺𝘰𝘶 use for building your flow cytometry panels? 

Stay tuned for 𝗗𝗮𝘆 𝟮𝟴: 𝗙𝗹𝗼𝘄 𝗖𝘆𝘁𝗼𝗺𝗲𝘁𝗿𝘆 𝗖𝗼𝗻𝘁𝗿𝗼𝗹𝘀 –𝗙𝗠𝗢, 𝗜𝘀𝗼𝘁𝘆𝗽𝗲, 𝗮𝗻𝗱 𝗘𝘅𝗽𝗲𝗿𝗶𝗺𝗲𝗻𝘁𝗮𝗹 𝗩𝗮𝗹𝗶𝗱𝗮𝘁𝗶𝗼𝗻 

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀 
1. https://www.cytometry.org/web/q_view.php?id=201&filter=Interpretation+and+Clinical+Application 
2. DOI: 10.1165/rcmb.2020-0149MA 
3. https://www.thermofisher.com/order/fluorescence-spectraviewer/#!/ 
4. https://www.ptglab.com/support/spectra-viewer/   

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