Re: Fixation of cells for TUNEL assay

Hi Mark

In those type of exps with an ethanol fix step, i've found that the 
first spin after the ethanol step is the most critical one in terms 
of subsets of cells not pelleting and being easy to lose. We commonly 
counteract that one by not spinning down in the ETOH purely and 
diluting the EtOH/cells at least 2 fold in PBS before that first 
quick spin. You always get some degree of loss but i dont believe 
we're anywhere near the 50% mark.

I also tend not to use aspirators or pipettes for supernatant 
removal, preferring the "gentle tip and tissue dab" method. I've 
always used this as I get concerned about vortices set up by 
aspirators and the like but that might just be me being OTT. I also 
always use the same tube as for running through the FACS, as 
unnecessary changeover seems to me to be just one more way to introduce loss.

Wash steps are usually at circa 800g (200-2400rpm in most standard 
bench top bucket centrifuges)  and the times for most steps well 
within the ranges you mentioned.
This works consistently well for us with many cell cycle assays which 
have a similar cell prep protocols .

  Paolo's point about different levels of "stickiness"	and loss 
between cell types is well made too. Have you seen this loss in all 
cell types or just some?

All the best

Matt


At 07:51 AM 3/13/06, Hollier, Mark J wrote:

>Hi everyone,
>
>I would like to thank the people who responded to my questions about 
>inducing apoptosis in PBMCs, preventing them from sticking to well 
>surfaces, and the information regarding two different populations of 
>live cells on FSC and SSC analysis.  I do have another question 
>though, and I hope someone can help me!
>
>During the fixation procedure for preparing peripheral blood 
>mononuclear cells (PBMCs) for a TUNEL assay (using BD Apodirect kit) 
>I get a loss of cells during the wash steps.  The cell number 
>decreases to approximately 50% of the original cells, a significant 
>cell loss.  I have tried using different centrifuge speeds (the BD 
>protocol suggests 300g, but I have also tried 400g, 600g, 800g, and 
>3800g) but they all give roughly the same cell loss.  I have also 
>tried different incubation times in the paraformaldehyde on ice (BD 
>protocol suggests 30-60 mins, I have tried 60, 90 or 120 mins) and 
>different incubation times in the 70% ethanol on ice before storage 
>at -20 C (BD protocol suggests 30 mins on ice or straight into the 
>freezer, I have tried 60, 90, 120, & 240 mins) for all possible 
>combinations.	However, no matter which variable I change, I still 
>keep getting similar percentages of cell loss.  I have also tried 
>different tubes for the fixation (15ml polypropylene Falcon tubes, 
>5ml polypropylene FACS tubes, and 1.5ml microcentrifuge tubes).
>
>Has anyone else experienced this cell loss?  Is it normal?  And if 
>not, what can I do to prevent it? Or what I am doing wrong? I do 
>carefully remove the supernatant by pipette after each 
>centrifugation, and (hopefully!) am not loosing cells via this mechanism.
>
>Many thanks to all who reply to this question, and hopefully there 
>is someone out there who can help me.
>
>Mark Hollier.
>
>Centers for Disease Control and Prevention,
>
>National Center for Infectious Diseases,
>
>Division Of Viral & Rickettsial Diseases,
>
>Viral Exanthems & Herpes Virus Branch,
>
>Atlanta, GA, USA
>
><mailto:etu4@cdc.gov>etu4@cdc.gov
>
>Tel: 404-639-2276
>
>Fax: 404-639-3540
>
>