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Cell number issue - Nile Red staining


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3 replies to this topic

#1 julnobody

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Posted 21 February 2013 - 01:50 PM

Hi,


I am doing Nile red staining on glioma cells,
- harvesting around 80000cells with 0,5% trypsin EDTA,
- inactivation with FBS, transfer to FACS tubes
- spinning 350g for 5mns,
- washing with 2mL PBS,
- spinning 350g for 5mns,
- adding 500uL Nile red working solution for 15mn
- spinning 350g for 5mns,
- washing in PBS 1x,
- respinning and finally resuspending in 200uL PBs for FACS analysis.

It is a lot of centrifugation steps which probably accounts for the fact that I have very few cells during FACS analysis, has anyone any other protocol that could enable recovering more cells ? (I'd like to add that I carefully remove the sup after each centrifugation step)


Many thanks

#2 Tabaluga

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Posted 21 February 2013 - 02:01 PM

Are there cells smeared against the wall of your reaction tube after centrifugation ?

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.

 


#3 julnobody

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Posted 22 February 2013 - 12:18 AM

Not that I can see but my pellet is usually hardly visible

#4 Tabaluga

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Posted 22 February 2013 - 08:09 AM

Is the pellet hardly visible from the start ? Because 80 000 cells is already a low number to start with, in my opinion.
Your protocol looks OK to me.

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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