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Short-term storage of HEK293 cells before FACS


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#1 Carnivora

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Posted 23 April 2013 - 01:17 AM

Hello all,

i am currently looking at the expression levels of a protein with an eGFP tag in HEK 293 cells via FACS analysis. For my next round of measurements i need 8h, 16h and 24h after induction.

Here is my Problem: in my experiments before (24h and 48h) i timed it in a way that i could do the FACS immediatly. But with these shorter times i will not be able to measure them immediatly.

So i am currently looking for a way to store my HEK293 cells for about two days that does not interfere with the FACS analysis and stops the cells from expressing any of my eGFP tagged protein.

Can i store them at 4°C or is -20°C preferable. Do i need to fixate them?

Any help is greatly appreciated.

Regards,

Edited by Carnivora, 23 April 2013 - 01:20 AM.


#2 cellthetruth

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Posted 23 April 2013 - 01:59 AM

don't store them if not necessary it may distort your results. if you really need to, compare stored/fresh (frozen/unfrozen) samples to be sure.
Anyways, you could fix them. some literature search would give you some interesting help/advice.
However, I would suggest you measure them immedeately. To do so you can simply induce the cells 24, 16, and 8 hours BEFORE measurements. this will save you the work of fixation. You need to be in the lab either way, to store, fix or induce the cells Posted Image

#3 Carnivora

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Posted 23 April 2013 - 06:11 AM

Thanks for the quick reply.
I found some literature about fixing cells with EtOH or PFA, but unfortunately you are right: no matter what i do, i will be in the lab for 24h straight... :-(
I think my best option at this point is to convince our lab tech to give me an in-deph introduction / trouble-shooting tips and let me use her facs on my own.

#4 Tabaluga

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Posted 24 April 2013 - 02:12 PM

If you have to store them, you have to fix them. However, I'm not sure about the effects this might have on the GFP fluorescence. As stated above, it may well distort your results.
If you have fixed the cells, they should be kept at 4°C (not -20° !) and in the dark, of course.

Edited by Tabaluga, 24 April 2013 - 02:15 PM.

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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