Re: detecting apoptosis in one cell type in coculture *vendor reply*

[Kilgore, Jason A.]

* 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
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at [phone]
[hidden email]
www.lifetechnologies.com

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----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

[Grant MacGregor]

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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.

Grant MacGregor


On Sep 9, 2015, at 11:50 AM, Kilgore, Jason <[hidden email]> wrote:

>
> * 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
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, then option 2.
> Or dial direct at [phone]
> [hidden email]
> www.lifetechnologies.com
>
> This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.
>
> -----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
>
> *****
> 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
>

[Kilgore, Jason A.]

*****
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.
*****

It certainly has been done, Grant, and published on.  

However, healthy cells also have phosphatidylserine exposure on their surface, labeled by annexin V, it's just that apoptotic cells have more.  Flow cytometers can easily distinguish the two populations, but in my experience it isn't as easy to distinguish the intensity differences via microscopy, due to the sensitivity of the detectors and our eyes and the lower sample sizes.  So I'd say proceed with caution.

Jason


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Grant MacGregor
Sent: Wednesday, September 09, 2015 6:18 PM
To: [hidden email]
Subject: Re: detecting apoptosis in one cell type in coculture *vendor reply*

*****
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.
*****

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.

Grant MacGregor


On Sep 9, 2015, at 11:50 AM, Kilgore, Jason <[hidden email]> wrote:

>
> * 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
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, then option 2.
> Or dial direct at [phone]
> [hidden email]
> www.lifetechnologies.com
>
> This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.
>
> -----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
>
> *****
> 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
>