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What should I suspend my cells in prior to cytometry?

DMEM no glucose no gl

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2 replies to this topic

#1 seanspotatobusiness

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Posted 28 March 2014 - 02:56 PM

I have been suspending my cells for flow cytometry in the regular medium I normally use sans FBS. I recently read that phenol red increases the background fluorescence for eGFP detection which is something I definitely don't want! I thought I could just go ahead and buy medium without phenol red in it but that also lacks glucose, glutamine-L and sodium pyruvate.

 

Are any of those needed for the cells to remain viable for about 90 minutes? How long are they okay in PBS? I just need them to not change in their spectral characteristics whilst they wait their turn on the flow cytometer. The fluorochromes in use are proteins expressed from genomic insertion and/or plasmids, fluorochrome-conjugated annexin V and a cell membrane-impermeant vitality stain (which I add 5-30 minutes prior to analysis).

 

If I go for one of the DMEMs, I think I might also add 1% FBS since I read that's tolerated by the cytometer and the cells remain viable for longer.

 

I've also been keeping my cells at room temperature and not on ice.

 

Options:

 

PBS (no calcium, no magnesium)

 

FluoroBrite™ DMEM for which the manufacturer suggests there is lower background than regular medium without phenol red - needs glutamine-L to be like my "normal" (I haven't done this long) cytometry medium.

 

DMEM, no glucose, no glutamine, no phenol red - needs glutamine-L, glucose and sodium pyruvate to be like my "normal" (I haven't done this long) cytometry medium.


Edited by seanspotatobusiness, 28 March 2014 - 02:59 PM.


#2 perneseblue

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Posted 30 March 2014 - 07:30 AM

I've only used PBS (no calcium, no magnesium) in ice. Cells are still viable after 30minutes. No idea if the cells are still alive after 90min. 

 

Lab tradition here says no FBS in the flow cytometer, Says it will clog up the machine and invite bacteria biofilm formation. Don't know if this is true or not.


May your PCR products be long, your protocols short and your boss on holiday

#3 Tabaluga

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Posted 30 March 2014 - 07:54 AM

We always used PBS with 1%BSA, others also used PBS with some FBS (forgot the percentage), on ice. At least some of our cells did survive more than 90 minutes because we also did long cell sorts with the cells suspended in PBS/1%BSA, although I should add that after the sorting they immediately came into contact with medium again.

Of course how much stress they tolerate is also dependent of the cell type. Also can't comment particularly on your stains.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 





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