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Removing cellular debris before flow cytometry


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#1 SuMi

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Posted 25 May 2009 - 03:10 AM

I have a sample which includes fluorescent bacteria, B cells, and cellular debris. I want to run the sample through the flow to determine the amount of fluorescence (and thus the presence of bacteria). I have adjusted the FSC and SSC settings for bacteria and so the B cells are not a problem. However, the debris is making up the majority of my events and so I am not getting a proper reading of fluoresence.

I wondered if anyone had any ideas as to how I could remove the debris while keeping bacteria. I can't pass my sample through a cell strainer as the debris is smaller than the bacteria and so this will also be filtered. Maybe a certain centrifuge speed?

Any help is greatly appreciated.

Thanks

#2 alan6017518

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Posted 28 May 2009 - 07:46 AM

I have a sample which includes fluorescent bacteria, B cells, and cellular debris. I want to run the sample through the flow to determine the amount of fluorescence (and thus the presence of bacteria). I have adjusted the FSC and SSC settings for bacteria and so the B cells are not a problem. However, the debris is making up the majority of my events and so I am not getting a proper reading of fluoresence.

I wondered if anyone had any ideas as to how I could remove the debris while keeping bacteria. I can't pass my sample through a cell strainer as the debris is smaller than the bacteria and so this will also be filtered. Maybe a certain centrifuge speed?

Any help is greatly appreciated.

Thanks


Not an expert in flow, but I will try my best,

I guess you can sort out the cells using both size selection and the florescence level, the size selection should get rid of the debris.

#3 Tdquerec

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Posted 11 June 2009 - 08:57 AM

I suggest staining for DNA to separate the bacteria from the debris. Search for publications and kits for flourescent/flow cytometry Gram-staining.

Edited by Tdquerec, 11 June 2009 - 09:31 AM.





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