Hello everyone,
I am trying to use a more accurate cell counting method than the traditional plate count method. Based on these two papers here,(http://www.ajstd.org/index.php/ajstd/article/view/495 and Article Flourishing deep-sea AAP bacteria detected by flow cytometri...
)one can use a cell sorter (e.g. FACS Aria or FACS Melody) to do this instead of a cell counter (e.g. FACS Accuri). However, their methods are not detailed enough for me to truly replicate it on our FACS Melody.
So my question is if anyone knows of a SOP for counting cells on cell sorters?
Thanks in advance!
Morgan
You could count on any of the FACS machines. The more advanced machines would of course be better. A few things you could try.
1. Contact the authors of the papers and ask if they would mind sharing detailed
protocols. Most people would be happy to.
2. The reasonably accurate method that is in use involves counting beads that several companies sell. You could follow the protocol that comes with it. There is even a thread on researchgate where someone has attached their calculation sheet.
https://www.google.com/amp/s/www.researchgate.net/post/Can_anyone_explain_about_the_procedure_for_true_count_beads_BD_Trucount_Tubes_in_counting_the_cells_by_flow_cytometry/amp
3. Contact BD and ask for help. They have in house people who specialize in counting cells/microbes and sub micron particles and they are really helpful. They can provide you with tips and protocols.
Julie Joseph, this was very helpful! The authors of the other papers used some sort of standard bead. Thank you for providing that link.
Hello Morgan, It's fair simple. The authors indicate they used DAPI, on cell filtered, fixed material. Cell were identified by their nuclear DNA labelling, under near-UV excitation. However, the Aria/Melody do NOT use syringe pumps, so don't give absolute cell counts. So the authours utilise a defined number of counting bead, added to fixed sample volume, to then extrapolate the cell count/ml (eg Polysciences Absolute Count standard, Biolegend Precision Count Beads, etc) based on the recorded bead numbers. If you do not have a near UV laser try 407nm excitation and 450nm emission, from your instrument. Best wishes
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