http://confocal-microscopy-list.275.s1.nabble.com/Re-detecting-apoptosis-in-one-cell-type-in-coculture-vendor-reply-tp7584203p7584204.html
As far as I am aware, fluorescently labelled Annexin V can be used to identify mammalian cells in the early stage of apoptosis using standard wide-field fluorescence microscopy.
>
> * VENDOR REPLY *
>
> Hi, Leoncio,
>
> I agree with Mike, with the approach, but I would suggest a different tracking dye to exclude the T cells. Calcein AM will be actively pumped out of the cells, sometimes within only an hour. So, instead, I would recommend labeling the T cells with CFDA SE or CellTracker Green CMFDA, which bind covalently to cytoplasmic proteins and won't be pumped out.
>
> I would also add that a flow cytometry-based system might also include an annexin V conjugate, though I'm guessing you are wanting a microscopy-based imaging analysis.
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes Tech Support
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of MODEL, MICHAEL
> Sent: Wednesday, September 09, 2015 11:09 AM
> To:
[hidden email]
> Subject: Re: detecting apoptosis in one cell type in coculture
>
> 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
>
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> 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
>