Re: P19 cell death

From: PLopez@adarc.org
Date: Mon Mar 25 2002 - 10:48:32 EST 

Hi Gene,

I have a couple of suggestions for your cell viability issue:

1- Is the CFSE bothering the cells? How is the viability if they are sorted
without the CFSE label?

2- How big are these cells? You may need to use an even larger nozzle than
the 100um you're using.

3- Can you chill everything down- cells, machine, collection media? That
may toughen up the cells.

4- What is the cell viability at the start? Cultures with low viability at
the start often don't do as well after sorting, even when using a live-cell
gate.

5 - I've heard it suggested that different buffers can react with each
other , such as carbonate buffers from the cell prep and the phosphate
buffered sheath.
You may want to try sorting with the cells in the same buffer as used on
the machine, with the addition of the FBS and antibiotics if you like. Use
the same thing for the collection media. Then after collection you can get
the cells  back into something they like.
A more expensive alternative is to run media as sheath.

6- I wonder if sorting with a larger sort-droplet envelope can help the
viability. I have no data on this,  but it's worth a shot.  You will not
get as many cells sorted do to aborts, but maybe it will help the viability
to have a few empty droplets sorted before and after the cell of interest.
Has anyone already looked into  this?


Peter Lopez
The Aaron Diamond AIDS Research Center
212.448.5188 (office)
212.448.5159 (fax)
212.448.5190 or 5110 (lab)



                      "Pizzo,Eugene"
                      <Pizzo@nso1.uchc.        To:       Cytometry Mailing List
                      edu>                      <cytometry@flowcyt.cyto.purdue.edu>
                                               cc:
                      03/21/2002 04:48         Subject:  P19 cell death
                      PM







Folks,

I'm seeing a fantastic amount of cell death following a low speed/low
pressure sort on my VantageSe and am wondering if anyone has experience
with these cells in particular or tips for preventing cell death in
general.
I'm getting about 10-20% live cell recovery following a typical low
pressure sort at 3K/sec,  8 psi, and 18khz with a 100um nozzle.
I normally get an average 70% recovery across the board.The cells are P19
embryonic carcinoma
cells with CFSE as a marker.  They are collected in DMEM with 20% FBS
and pen/strep.
Thanks.

Gene Pizzo/UCONN Health

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