Re: FITC/PI stain whole fixed cells
Julie Auger (jauger@flowcity.bsd.uchicago.edu)
Thu, 28 Sep 1995 16:14:28 -0500
>To: mclement@milbrandt.wustl.edu (Mark Clements)
>From: jauger@flowcity.bsd.uchicago.edu (Julie Auger)
>Subject: Re: FITC/PI stain whole fixed cells
>Cc: cytometry
>Bcc:
>X-Attachments:
>
>Mark -
>try fixing in less volume and a final concentration of 70% EtOH i.e.
resuspend in 1.3ml PBS and add 3.0ml EtOH in a 15ml conical. This is good
for up to 3-4 million cells - double for more. After your 1hr fixation,
bring the volume up to 15ml w/ PBS to wash. Then try spinning HARDER after
fixation. The density of the cells changes after fixation and they are
harder to pellet. Also diluting out the EtOH with PBS will help, as cells
do not pellet well in EtOH. Don't spin too hard 2200-2300 rpm should be fine.
>
>Hope this helps
>Julie
>
>
>>I'm relatively new to flow technology, and I'm experiencing the following
>>technical difficulty:
>>
>> I want to dual-stain cells of the osteosarcoma cell line SAOS-2 with
>>FITC/PI. The FITC-conjugated antibody is against a transfected surface
>>antigen for which I am achieving a transfection efficiency in the
>>neighborhood of 3-5% The only problem is that I keep losing a significant
>>percentage of the cells during the preparation protocol, presumably due to
>>damage/lysis. Normally, I harvest the cells (plated at 250K per 10cm plate)
>>with 0.1% EDTA in PBS for 10-15', spin @2K rpm for 5', incubate with 20 ul
>>FITC antibody in 80 ul media for 30', rinse in PBS + 1% FCS and spin @2K rpm
>>for 5', resuspend in 1.5 ml PBS, add 13.5 ml EtOH, fix @4'C for at least
>>1hr, spin @1.5K rpm for 5', and resuspend in PI stain (PI + RNAse + PBS + 1%
>>FCS).
>> Using this protocol, I usually lose about 60-70% of my cells between
>> harvesting to PI staining. I know that protocols such as this are commonly
>>used; I just haven't been able to figure out why I'm losing so many cells!
>>This loss is significant, because I need about 300,000 cells in order to
>>obtain enough cells positive for the surface antigen to allow DNA analysis.
>>The only thing I can think to do right now is use larger plates and more
>>cells for my
>>transfections, but I'm not still not comfortable with that high rate of cell
>>loss. Any suggestions or better protocols out there? Anybody trying similar
>>things with osteosarcoma cells?
>>
>>Mark Clements
>>mclement@milbrandt.wustl.edu
>>
>>
>>
>
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
