Cell number issue - Nile Red staining - (Feb/21/2013 )

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

-julnobody-

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

-Tabaluga-

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

-julnobody-

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.

-Tabaluga-