2 replies to this topic

[SuMi]

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

[alan6017518]

SuMi, on May 25 2009, 04:10 AM, said:

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.

[Tdquerec]

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.