Re: Summary of responses to fixation of cells and cell loss during wash steps

Hi all,

I would just like to thank everyone who replied about my question
regarding the loss of cells during the fixation and/or wash steps of the
TUNEL assay.  Below is a summary of the responses I got, as several
people replied saying they had similar problems and wanted to know any
possible solutions.  Thankfully the problem was mostly solved, to an
acceptable level anyway, by your suggestions.

1.	Add BSA to the wash buffer.  Recommended percentages (w/v) were
0.1%, 1%, and 2%.  This was the first variable I tried changing, and it
seemed to solve most of the problem.  I still did loose some cells,
mainly after removing the ethanol (approximately 15-30%).  But after
each wash with PBS/1% BSA I only lost approximately 10% more cells per
wash.  When the washes were performed with PBS alone I lost
approximately 50-60% of the cells per wash.

2.	To decant the wash buffer (tip and tissue dab), instead of
aspiration.

3.	Use swing buckets in the centrifugation rather than fixed angle
rotors.

4.	Increase the starting number of cells.	Some people replied that
it was expected to loose 50% of your cells during the wash steps and to
increase the initial cell number so that you end up with the correct
number of cells.

5.	A couple of responses suggested 15ml polypropylene (Falcon)
tubes gave less cell loss than with the 5ml polystyrene (FACS) tubes or
microcentrifuge tubes.

6.	Increase centrifuge speed from 300g to 800-1000g during the wash
steps.

7.	Centrifuge for longer times, suggestions included 10, 15, and 30
mins.

8.	Reduce the number of wash steps, but this may cause higher
background readings later.

9.	Use a one step fix/perm process instead of the two step
paraformaldehyde/ethanol.  However, having spoken with the technical
support at BD Pharmingen about using their cytofix/cytoperm kit they
said that once the cells were washed in PBS the cells would revert to
being non-permeabilized (as the method is reversible with that kit).
This would prevent the end-labelling in the TUNEL assay.  If the wash
buffer provided with the kit was used instead of PBS, then the labelling
reaction would be prevented due to the presence of residual wash buffer.
Does anyone have more information about this?  Or have tried this?



I think another point was made by Zbigniew Darzynkiewicz:

Hello Mark and Mat,

We have measured cell loss after centrifugations in the presence and
absence of bovine serum (Bedner at al., Cytometry,29:191-196,1997; see
Table 1 in this article). Starting with 1 x 10E6  PBC per 1 ml,
consisting of 48% lymphs, 8% monocytes and 44% granulocytes after 7
repeated centrifugations in PBS alone we obtained 0.08 x 10E6 cells per
1 ml consisting 71% lymphs, 2% monocytes and 27% granulocytes. When the
cells were centrifuged while suspended in PBS containing 10% (v/v)
bovine serum, after 7 centrifugations there were 0.24 x 10E6 cells per 1
ml, consisting of 58% lymphs, 6% monocytes and 36% granulocytes.  Thus,
the presence of bovine serum definitely reduced cell loss. However, the
proportions of cells were changed - the most "sticky" were granulocytes,
while the least - lymphocytes.  

Zbigniew Darzynkiewicz, M.D., Ph.D.
Brander Cancer Research Institute at NYMC
19 Bradhurst Avenue, Suite 2400
Hawthorne, N.Y. 10532
darzynk@nymc.edu
http://www.darzynkiewicz.com/zbigniew/


Once again, i would like to thank everyone who responded to my plea for
help with the cell loss,

Mark Hollier.



Dr 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
E-mail:etu4@cdc.gov
Tel: 404-639-2276
Fax: 404-639-3540