Re: detecting apoptosis in one cell type in coculture

Posted by mmodel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/detecting-apoptosis-in-one-cell-type-in-coculture-tp7584201p7584202.html

Hi Leoncio

Apoptosis is usually not decided by a single assay but by a combination of several assays. Besides, apoptotic cells can sometimes detach, depending on the cell type and the stimulus. My first thought is to label T cells with a fluorescent marker (calcein), then trypsinize everything and analyze those that are calcein-negative using, for example, DNA aggregation (blue), depolarization of mitochondria (red) and maybe a far-red assay for caspase. Or if you can use a confocal system then T cells sitting on top shouldn't be an issue

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Leoncio Vergara
Sent: Wednesday, September 09, 2015 12:31 PM
To: [hidden email]
Subject: detecting apoptosis in one cell type in coculture

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Good morning

We are working on setting up an image screening assay in which we want to measure apoptosis in one cell type (adherent)  after exposure to T cells (non adherent). the adherent cells are grown on a monolayer and the T cells are added on top and incubated for a few hrs (~3-5 hrs). We want to study apoptosis in the adherent cells without interference from the T cells.
CellEvent (Thermo Fisher) works great in other conditions and is ideal for a screening assay, but in this case it does not work because of the presence of the Tcells, We would like to know is there is an alternative indicator we can load in the adherent cells and wash prior to the T cells.
We need to test multiple cell lines so fluorescent protein based indicators would not be ideal.

Thanks in advance for your help.

Leoncio Vergara MD
Co-Director

Center for Advanced Imaging (CAI) at the

Center for Translational Cancer Research (CTCR),

Institute for Biosciences and Technology,

Texas A&M Health Sciences Center,

Houston, Texas 77030