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"Gentle" Live Cell Dye for Primary Cells

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Cameron Nowell

"Gentle" Live Cell Dye for Primary Cells

Hi List,

We are currently trying to do some live imaging of tube formation with primary cells. It works great under DIC/Phase but we want to be able to fluorescently tag them so we can measure some kinetics of what is going on. We have tried CellTracker and Calcein so far with very little success. The cells pretty much explode after about 12 hours and don’t form very good (or any) tubes. Celltracker lets tubes form but there is more cell death than in the DIC/Phase alone capture. Calcein made everything explode (quite pretty but not very useful).

Does anyone have any suggestions on a dye or culture media additives that might stop/reduce this happening? We have used these dyes in the past on cell lines but primary cells seem a bit more fragile. We have tried LavaCell in the past and found it to be rather unstable and bleach quickly.

The imaging is being carried out on a widefield scope with a 37 degree incubator on it and 5% CO2 being supplied. Acquisition is once every 30 minutes for 24 hours, with exposures less than 500ms using a fast shutter (so fluorescent exposure is being kept to a minimum).

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

Facility Website

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

Cameron Nowell

Re: "Gentle" Live Cell Dye for Primary Cells

Hi Again,

To add a little bit more information.

- The CellaTracker is CellTracker Green

- Calcein is Calcein AM

- Light source is a mercury lamp (103W) attached directly to the microscope

- Camera is a SPOT Pursuit

Cheers

Cam

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Thursday, 20 May 2010 8:55 AM
To: [hidden email]
Subject: "Gentle" Live Cell Dye for Primary Cells

Hi List,

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

Facility Website

Tim Feinstein-2

Re: "Gentle" Live Cell Dye for Primary Cells

<base href="x-msg://25/">Hi Cameron,

You might want to try chloromethylfluorescein diacetate (CMFDA). Alternatively, add a reagent like Trolox (~300 micromolar would work) to test whether photooxidation is causing your problem.

good luck,

Tim

Timothy Feinstein, PhD

Postdoctoral Associate, Vilardaga lab

University of Pittsburgh Dept. of Pharmacology

Pittsburgh, PA, USA

On May 19, 2010, at 7:28 PM, Cameron Nowell wrote:

Hi Again,

To add a little bit more information.

-          The CellaTracker is CellTracker Green
-          Calcein is Calcein AM
-          Light source is a mercury lamp (103W) attached directly to the microscope
-          Camera is a SPOT Pursuit

Cheers

Cam

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Thursday, 20 May 2010 8:55 AM
To: [hidden email]
Subject: "Gentle" Live Cell Dye for Primary Cells

Hi List,

We are currently trying to do some live imaging of tube formation with primary cells. It works great under DIC/Phase but we want to be able to fluorescently tag them so we can measure some kinetics of what is going on. We have tried CellTracker and Calcein so far with very little success. The cells pretty much explode after about 12 hours and don’t form very good (or any) tubes. Celltracker lets tubes form but there is more cell death than in the DIC/Phase alone capture. Calcein made everything explode (quite pretty but not very useful).

Does anyone have any suggestions on a dye or culture media additives that might stop/reduce this happening? We have used these dyes in the past on cell lines but primary cells seem a bit more fragile. We have tried LavaCell in the past and found it to be rather unstable and bleach quickly.

The imaging is being carried out on a widefield scope with a 37 degree incubator on it and 5% CO2 being supplied. Acquisition is once every 30 minutes for 24 hours, with exposures less than 500ms using a fast shutter (so fluorescent exposure is being kept to a minimum).

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

Julio Vazquez

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Cameron Nowell

One issue I have seen is fluorescence filter sets leaking UV, perhaps specially (but not exclusively) as they get older.... If you don't already have one, I would recommend getting an additional UV blocking filter and inserting it in the excitation light path.

Also, the recommended concentrations for cell dyes are sometimes much higher than needed.... try using less dye and see what happens...

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA

http://www.fhcrc.org/

On May 19, 2010, at 4:28 PM, Cameron Nowell wrote:

Hi Again,

To add a little bit more information.

-          The CellaTracker is CellTracker Green
-          Calcein is Calcein AM
-          Light source is a mercury lamp (103W) attached directly to the microscope
-          Camera is a SPOT Pursuit

Cheers

Cam

From: Confocal Microscopy List [[hidden email]] On Behalf Of Cameron Nowell
Sent: Thursday, 20 May 2010 8:55 AM
To: [hidden email]
Subject: "Gentle" Live Cell Dye for Primary Cells

Hi List,

We are currently trying to do some live imaging of tube formation with primary cells. It works great under DIC/Phase but we want to be able to fluorescently tag them so we can measure some kinetics of what is going on. We have tried CellTracker and Calcein so far with very little success. The cells pretty much explode after about 12 hours and don’t form very good (or any) tubes. Celltracker lets tubes form but there is more cell death than in the DIC/Phase alone capture. Calcein made everything explode (quite pretty but not very useful).

Does anyone have any suggestions on a dye or culture media additives that might stop/reduce this happening? We have used these dyes in the past on cell lines but primary cells seem a bit more fragile. We have tried LavaCell in the past and found it to be rather unstable and bleach quickly.

The imaging is being carried out on a widefield scope with a 37 degree incubator on it and 5% CO2 being supplied. Acquisition is once every 30 minutes for 24 hours, with exposures less than 500ms using a fast shutter (so fluorescent exposure is being kept to a minimum).

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

Dale Callaham

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Cameron Nowell

Hi Cam,

Do the cells explode even if not observed? Is it the combination of the
dye, then the exposures, or just toxicity of the dyes? I know that it
isn't easy to add hardware, but I am hearing that a train rapid pulses
from the LED sources capable of such modulation is far less damaging to
cells than exposure to the arc lamp. I do not have one of these systems
(sadly the "stimulus funds" have missed me) and have no direct
experience, but maybe someone will chime in and confirm/deny this rumor.

Dale

Cameron Nowell wrote:

>
>
> Hi Again,
>
> To add a little bit more information.
>
> - The CellaTracker is CellTracker Green
>
> - Calcein is Calcein AM
>
> - Light source is a mercury lamp (103W) attached directly to the microscope
>
> - Camera is a SPOT Pursuit
>
> Cheers
>
> Cam
>
> *From:* Confocal Microscopy List
> [mailto:[hidden email]] *On Behalf Of *Cameron Nowell
> *Sent:* Thursday, 20 May 2010 8:55 AM
> *To:* [hidden email]
> *Subject:* "Gentle" Live Cell Dye for Primary Cells
>
> Hi List,
>
> We are currently trying to do some live imaging of tube formation with
> primary cells. It works great under DIC/Phase but we want to be able to
> fluorescently tag them so we can measure some kinetics of what is going
> on. We have tried CellTracker and Calcein so far with very little
> success. The cells pretty much explode after about 12 hours and don’t
> form very good (or any) tubes. Celltracker lets tubes form but there is
> more cell death than in the DIC/Phase alone capture. Calcein made
> everything explode (quite pretty but not very useful).
>
> Does anyone have any suggestions on a dye or culture media additives
> that might stop/reduce this happening? We have used these dyes in the
> past on cell lines but primary cells seem a bit more fragile. We have
> tried LavaCell in the past and found it to be rather unstable and bleach
> quickly.
>
> The imaging is being carried out on a widefield scope with a 37 degree
> incubator on it and 5% CO2 being supplied. Acquisition is once every 30
> minutes for 24 hours, with exposures less than 500ms using a fast
> shutter (so fluorescent exposure is being kept to a minimum).
>
> Thanks
>
> Cam
>
> *Cameron J. Nowell
> *Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> *Office*: +61 3 9341 3155
> *Mobile*: +61422882700
> *Fax*: +61 3 9341 3104
>
> Facility Website <http://www.ludwig.edu.au/confocal/>
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>

Cameron Nowell

Re: "Gentle" Live Cell Dye for Primary Cells

Hi Dale,

The calcein stained cells exploded outside the captured field (though not as badly it seems). With the cell tracker there wasn't much difference either so i am guessing they just don't like the dye.

Cam

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Callaham
Sent: Thursday, 20 May 2010 1:46 PM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

Hi Cam,

Do the cells explode even if not observed? Is it the combination of the
dye, then the exposures, or just toxicity of the dyes? I know that it
isn't easy to add hardware, but I am hearing that a train rapid pulses
from the LED sources capable of such modulation is far less damaging to
cells than exposure to the arc lamp. I do not have one of these systems
(sadly the "stimulus funds" have missed me) and have no direct
experience, but maybe someone will chime in and confirm/deny this rumor.

Dale

Cameron Nowell wrote:

Axel Kurt Preuss

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Dale Callaham

I would just try the Cell IQ Chipman. No need for dyes. Long term observation , cell recognition by machine vision, and LED on only for exposure.

I ve no commercial interest

Thank you and best regards

Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging IMCB AStar , Singapore

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Callaham
Sent: Thursday, May 20, 2010 11:46 AM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

Hi Cam,

Do the cells explode even if not observed? Is it the combination of the
dye, then the exposures, or just toxicity of the dyes? I know that it
isn't easy to add hardware, but I am hearing that a train rapid pulses
from the LED sources capable of such modulation is far less damaging to
cells than exposure to the arc lamp. I do not have one of these systems
(sadly the "stimulus funds" have missed me) and have no direct
experience, but maybe someone will chime in and confirm/deny this rumor.

Dale

Cameron Nowell wrote:

>
>
> Hi Again,
>
> To add a little bit more information.
>
> - The CellaTracker is CellTracker Green
>
> - Calcein is Calcein AM
>
> - Light source is a mercury lamp (103W) attached directly to the microscope
>
> - Camera is a SPOT Pursuit
>
> Cheers
>
> Cam
>
> *From:* Confocal Microscopy List
> [mailto:[hidden email]] *On Behalf Of *Cameron Nowell
> *Sent:* Thursday, 20 May 2010 8:55 AM
> *To:* [hidden email]
> *Subject:* "Gentle" Live Cell Dye for Primary Cells
>
> Hi List,
>
> We are currently trying to do some live imaging of tube formation with
> primary cells. It works great under DIC/Phase but we want to be able to
> fluorescently tag them so we can measure some kinetics of what is going
> on. We have tried CellTracker and Calcein so far with very little
> success. The cells pretty much explode after about 12 hours and don't
> form very good (or any) tubes. Celltracker lets tubes form but there is
> more cell death than in the DIC/Phase alone capture. Calcein made
> everything explode (quite pretty but not very useful).
>
> Does anyone have any suggestions on a dye or culture media additives
> that might stop/reduce this happening? We have used these dyes in the
> past on cell lines but primary cells seem a bit more fragile. We have
> tried LavaCell in the past and found it to be rather unstable and bleach
> quickly.
>
> The imaging is being carried out on a widefield scope with a 37 degree
> incubator on it and 5% CO2 being supplied. Acquisition is once every 30
> minutes for 24 hours, with exposures less than 500ms using a fast
> shutter (so fluorescent exposure is being kept to a minimum).
>
> Thanks
>
> Cam
>
> *Cameron J. Nowell
> *Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> *Office*: +61 3 9341 3155
> *Mobile*: +61422882700
> *Fax*: +61 3 9341 3104
>
> Facility Website <http://www.ludwig.edu.au/confocal/>
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Rosemary.White

Re: "Gentle" Live Cell Dye for Primary Cells

Hi Cam,

Not sure what your tubes are, but in plants, cell components can explode if
they're overloaded with dye. For example, if you load up with one of the
carboxycyanine dyes, e.g. DiOC6 for ER, if you overdo it, the mitochondria
light up brightly, then when the ER lights up, the mitochondria explode.
Using low concentration, and rinsing off even before you can see any
fluorescence solves this problem.
cheers,
Rosemary

On 20/05/10 6:51 PM, "Axel Kurt Preuss" <[hidden email]> wrote:

> I would just try the Cell IQ Chipman. No need for dyes. Long term
> observation , cell recognition by machine vision, and LED on only for
> exposure.
>
> I ve no commercial interest
>
> Thank you and best regards
>
> Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging
> IMCB AStar , Singapore
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Dale Callaham
> Sent: Thursday, May 20, 2010 11:46 AM
> To: [hidden email]
> Subject: Re: "Gentle" Live Cell Dye for Primary Cells
>
> Hi Cam,
>
> Do the cells explode even if not observed? Is it the combination of the
> dye, then the exposures, or just toxicity of the dyes? I know that it
> isn't easy to add hardware, but I am hearing that a train rapid pulses
> from the LED sources capable of such modulation is far less damaging to
> cells than exposure to the arc lamp. I do not have one of these systems
> (sadly the "stimulus funds" have missed me) and have no direct
> experience, but maybe someone will chime in and confirm/deny this rumor.
>
> Dale
>
> Cameron Nowell wrote:
>>
>>
>> Hi Again,
>>
>> To add a little bit more information.
>>
>> - The CellaTracker is CellTracker Green
>>
>> - Calcein is Calcein AM
>>
>> - Light source is a mercury lamp (103W) attached directly to the microscope
>>
>> - Camera is a SPOT Pursuit
>>
>> Cheers
>>
>> Cam
>>
>> *From:* Confocal Microscopy List
>> [mailto:[hidden email]] *On Behalf Of *Cameron Nowell
>> *Sent:* Thursday, 20 May 2010 8:55 AM
>> *To:* [hidden email]
>> *Subject:* "Gentle" Live Cell Dye for Primary Cells
>>
>> Hi List,
>>
>> We are currently trying to do some live imaging of tube formation with
>> primary cells. It works great under DIC/Phase but we want to be able to
>> fluorescently tag them so we can measure some kinetics of what is going
>> on. We have tried CellTracker and Calcein so far with very little
>> success. The cells pretty much explode after about 12 hours and don't
>> form very good (or any) tubes. Celltracker lets tubes form but there is
>> more cell death than in the DIC/Phase alone capture. Calcein made
>> everything explode (quite pretty but not very useful).
>>
>> Does anyone have any suggestions on a dye or culture media additives
>> that might stop/reduce this happening? We have used these dyes in the
>> past on cell lines but primary cells seem a bit more fragile. We have
>> tried LavaCell in the past and found it to be rather unstable and bleach
>> quickly.
>>
>> The imaging is being carried out on a widefield scope with a 37 degree
>> incubator on it and 5% CO2 being supplied. Acquisition is once every 30
>> minutes for 24 hours, with exposures less than 500ms using a fast
>> shutter (so fluorescent exposure is being kept to a minimum).
>>
>> Thanks
>>
>> Cam
>>
>> *Cameron J. Nowell
>> *Microscopy Manager
>> Centre for Advanced Microscopy
>> Ludwig Institute for Cancer Research
>> PO Box 2008
>> Royal Melbourne Hospital
>> Victoria, 3050
>> AUSTRALIA
>>
>> *Office*: +61 3 9341 3155
>> *Mobile*: +61422882700
>> *Fax*: +61 3 9341 3104
>>
>> Facility Website <http://www.ludwig.edu.au/confocal/>
>>
>> This communication is intended only for the named recipient and may
>> contain information that is confidential, legally privileged or subject
>> to copyright; the Ludwig Institute for Cancer Research Ltd does not
>> waive any rights if you have received this communication in error.
>> The views expressed in this communication are those of the sender and do
>> not necessarily reflect the views of the Ludwig Institute for Cancer
>> Research Ltd.
>>
>> This communication is intended only for the named recipient and may
>> contain information that is confidential, legally privileged or subject
>> to copyright; the Ludwig Institute for Cancer Research Ltd does not
>> waive any rights if you have received this communication in error.
>> The views expressed in this communication are those of the sender and do
>> not necessarily reflect the views of the Ludwig Institute for Cancer
>> Research Ltd.
>>
>
> Note: This message may contain confidential information. If this Email/Fax has
> been sent to you by mistake, please notify the sender and delete it
> immediately. Thank you.

Axel Kurt Preuss

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Axel Kurt Preuss

Dear Cam
One way , as I previously posted, is to avoid dyes and use machine vision for recognizing certain cell types

The other way would be to try membrane dyes, or syto live stains at very low concentrations, or a ph dye just for visualizing the cells. Any long wavelength dye with excitation not lower than 488 and not higher than 562

You probably will have to keep serum in the culture. DMEM F12 with certain lots of FCS. Sometimes a cocktail of additives helps. But then again, you might need more antibiotics.

Good to know would be how unstained cells behave under Hg light exposing conditions used for stained cells.

Keeping low Ca (300uM) external might help, and keeping radicals at bay as suggested by some.

In the end, avoidance of dyes is the best way to go

Thank you and best regards

Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging IMCB

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Axel Kurt Preuss
Sent: Thursday, May 20, 2010 4:52 PM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

I would just try the Cell IQ Chipman. No need for dyes. Long term observation , cell recognition by machine vision, and LED on only for exposure.

I ve no commercial interest

Thank you and best regards

Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging IMCB AStar , Singapore

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Callaham
Sent: Thursday, May 20, 2010 11:46 AM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

Hi Cam,

Do the cells explode even if not observed? Is it the combination of the
dye, then the exposures, or just toxicity of the dyes? I know that it
isn't easy to add hardware, but I am hearing that a train rapid pulses
from the LED sources capable of such modulation is far less damaging to
cells than exposure to the arc lamp. I do not have one of these systems
(sadly the "stimulus funds" have missed me) and have no direct
experience, but maybe someone will chime in and confirm/deny this rumor.

Dale

Cameron Nowell wrote:

>
>
> Hi Again,
>
> To add a little bit more information.
>
> - The CellaTracker is CellTracker Green
>
> - Calcein is Calcein AM
>
> - Light source is a mercury lamp (103W) attached directly to the microscope
>
> - Camera is a SPOT Pursuit
>
> Cheers
>
> Cam
>
> *From:* Confocal Microscopy List
> [mailto:[hidden email]] *On Behalf Of *Cameron Nowell
> *Sent:* Thursday, 20 May 2010 8:55 AM
> *To:* [hidden email]
> *Subject:* "Gentle" Live Cell Dye for Primary Cells
>
> Hi List,
>
> We are currently trying to do some live imaging of tube formation with
> primary cells. It works great under DIC/Phase but we want to be able to
> fluorescently tag them so we can measure some kinetics of what is going
> on. We have tried CellTracker and Calcein so far with very little
> success. The cells pretty much explode after about 12 hours and don't
> form very good (or any) tubes. Celltracker lets tubes form but there is
> more cell death than in the DIC/Phase alone capture. Calcein made
> everything explode (quite pretty but not very useful).
>
> Does anyone have any suggestions on a dye or culture media additives
> that might stop/reduce this happening? We have used these dyes in the
> past on cell lines but primary cells seem a bit more fragile. We have
> tried LavaCell in the past and found it to be rather unstable and bleach
> quickly.
>
> The imaging is being carried out on a widefield scope with a 37 degree
> incubator on it and 5% CO2 being supplied. Acquisition is once every 30
> minutes for 24 hours, with exposures less than 500ms using a fast
> shutter (so fluorescent exposure is being kept to a minimum).
>
> Thanks
>
> Cam
>
> *Cameron J. Nowell
> *Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> *Office*: +61 3 9341 3155
> *Mobile*: +61422882700
> *Fax*: +61 3 9341 3104
>
> Facility Website <http://www.ludwig.edu.au/confocal/>
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Cameron Nowell

Re: "Gentle" Live Cell Dye for Primary Cells

Hi List,

Thanks for all the suggestions so far i will look into some of the other dyes and also try lowering the concentration of the existing dyes.

Axel - the machine recognition idea will not work for this situation. I don't need to count the cells i need to be able to light up the whole cell/tube network as it forms

http://www.bdbiosciences.com/cellculture/endothelialcells/index.jsp#

the link above shows some images of tube formation that are similar to what we get (our tubes tend to be a bit fatter as the assay uses lymphatic cells).

We already have a very robust and advanced analysis method for end point (ie fixed and stained tubes after 24 hours). But we want to be able to measure the kinetics of the formation (tube length, node number, node area, set connection etc) by applying this analysis to a time aquisition. To do this we need a stable , non-interfereing cell dye that will work in primary cells.

Membrane dyes are not an option as they turn over wasy to fast (like in 10 minutes). Ideally a stable cell line exprssing GFP (or similar) woudl be best but unfortunately is not an option in this case.

Cheers

Cam

________________________________

From: Confocal Microscopy List on behalf of Axel Kurt Preuss
Sent: Thu 20/05/2010 7:06 PM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

Dear Cam
One way , as I previously posted, is to avoid dyes and use machine vision for recognizing certain cell types

The other way would be to try membrane dyes, or syto live stains at very low concentrations, or a ph dye just for visualizing the cells. Any long wavelength dye with excitation not lower than 488 and not higher than 562

You probably will have to keep serum in the culture. DMEM F12 with certain lots of FCS. Sometimes a cocktail of additives helps. But then again, you might need more antibiotics.

Good to know would be how unstained cells behave under Hg light exposing conditions used for stained cells.

Keeping low Ca (300uM) external might help, and keeping radicals at bay as suggested by some.

In the end, avoidance of dyes is the best way to go

Thank you and best regards

Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging IMCB

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Axel Kurt Preuss
Sent: Thursday, May 20, 2010 4:52 PM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

I would just try the Cell IQ Chipman. No need for dyes. Long term observation , cell recognition by machine vision, and LED on only for exposure.

I ve no commercial interest

Thank you and best regards

Axel "We focus on your objectives!" -Axel K Preuss PhD, Central Imaging IMCB AStar , Singapore

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Callaham
Sent: Thursday, May 20, 2010 11:46 AM
To: [hidden email]
Subject: Re: "Gentle" Live Cell Dye for Primary Cells

Hi Cam,

Do the cells explode even if not observed? Is it the combination of the
dye, then the exposures, or just toxicity of the dyes? I know that it
isn't easy to add hardware, but I am hearing that a train rapid pulses
from the LED sources capable of such modulation is far less damaging to
cells than exposure to the arc lamp. I do not have one of these systems
(sadly the "stimulus funds" have missed me) and have no direct
experience, but maybe someone will chime in and confirm/deny this rumor.

Dale

Cameron Nowell wrote:

>
>
> Hi Again,
>
> To add a little bit more information.
>
> - The CellaTracker is CellTracker Green
>
> - Calcein is Calcein AM
>
> - Light source is a mercury lamp (103W) attached directly to the microscope
>
> - Camera is a SPOT Pursuit
>
> Cheers
>
> Cam
>
> *From:* Confocal Microscopy List
> [mailto:[hidden email]] *On Behalf Of *Cameron Nowell
> *Sent:* Thursday, 20 May 2010 8:55 AM
> *To:* [hidden email]
> *Subject:* "Gentle" Live Cell Dye for Primary Cells
>
> Hi List,
>
> We are currently trying to do some live imaging of tube formation with
> primary cells. It works great under DIC/Phase but we want to be able to
> fluorescently tag them so we can measure some kinetics of what is going
> on. We have tried CellTracker and Calcein so far with very little
> success. The cells pretty much explode after about 12 hours and don't
> form very good (or any) tubes. Celltracker lets tubes form but there is
> more cell death than in the DIC/Phase alone capture. Calcein made
> everything explode (quite pretty but not very useful).
>
> Does anyone have any suggestions on a dye or culture media additives
> that might stop/reduce this happening? We have used these dyes in the
> past on cell lines but primary cells seem a bit more fragile. We have
> tried LavaCell in the past and found it to be rather unstable and bleach
> quickly.
>
> The imaging is being carried out on a widefield scope with a 37 degree
> incubator on it and 5% CO2 being supplied. Acquisition is once every 30
> minutes for 24 hours, with exposures less than 500ms using a fast
> shutter (so fluorescent exposure is being kept to a minimum).
>
> Thanks
>
> Cam
>
> *Cameron J. Nowell
> *Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> *Office*: +61 3 9341 3155
> *Mobile*: +61422882700
> *Fax*: +61 3 9341 3104
>
> Facility Website <http://www.ludwig.edu.au/confocal/>
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or subject
> to copyright; the Ludwig Institute for Cancer Research Ltd does not
> waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do
> not necessarily reflect the views of the Ludwig Institute for Cancer
> Research Ltd.
>

Steffen Dietzel

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Julio Vazquez

I'd second Julio's line of reasoning. If you say cells outside the
field of view explode too but not as badly, you might have a mixed problem.

Things you could try:
- image te cells with the mercury lamp without a dye and see if they survive.
- image them twice/4x as often: do they now explode already after 6/3 h?
- on many scopes, you can exchange the lamp house of the mercury lamp
with the lamp house of the halogen 12 V lamp. Try using the halogen
12 V for fluorescence excitation. The image will be only ~ one third
as bright and exposure times respectively longer. But it didn't sound
like you were in a hurry. 12 V lamps emit very little UV, compared
to mercury bulbs. Extra UV blockers wouldn't hurt though. Of
coursce, whether or not the UV dose is a problem also depends on your
objective magnification, i.e. the size of the specimen on which you
distribute the given UV light.

Steffen

At 02:36 20.05.2010, you wrote:

>One issue I have seen is fluorescence filter sets leaking UV,
>perhaps specially (but not exclusively) as they get older.... If you
>don't already have one, I would recommend getting an additional UV
>blocking filter and inserting it in the excitation light path.
>
>Also, the recommended concentrations for cell dyes are sometimes
>much higher than needed.... try using less dye and see what happens...
>
>
>--
>Julio Vazquez
>Fred Hutchinson Cancer Research Center
>Seattle, WA
>
><http://www.fhcrc.org>http://www.fhcrc.org/
>
>
>On May 19, 2010, at 4:28 PM, Cameron Nowell wrote:
>
>>Hi Again,
>>
>>To add a little bit more information.
>>
>>- The CellaTracker is CellTracker Green
>>- Calcein is Calcein AM
>>- Light source is a mercury lamp (103W) attached directly
>>to the microscope
>>- Camera is a SPOT Pursuit
>>
>>
>>Cheers
>>
>>Cam
>>
>>
>>
>>From: Confocal Microscopy List
>>[<mailto:[hidden email]>mailto:[hidden email]]
>>On Behalf Of Cameron Nowell
>>Sent: Thursday, 20 May 2010 8:55 AM
>>To: <mailto:[hidden email]>[hidden email]
>>Subject: "Gentle" Live Cell Dye for Primary Cells
>>
>>Hi List,
>>
>>We are currently trying to do some live imaging of tube formation
>>with primary cells. It works great under DIC/Phase but we want to
>>be able to fluorescently tag them so we can measure some kinetics
>>of what is going on. We have tried CellTracker and Calcein so far
>>with very little success. The cells pretty much explode after about
>>12 hours and don't form very good (or any) tubes. Celltracker lets
>>tubes form but there is more cell death than in the DIC/Phase alone
>>capture. Calcein made everything explode (quite pretty but not very useful).
>>
>>Does anyone have any suggestions on a dye or culture media
>>additives that might stop/reduce this happening? We have used these
>>dyes in the past on cell lines but primary cells seem a bit more
>>fragile. We have tried LavaCell in the past and found it to be
>>rather unstable and bleach quickly.
>>
>>The imaging is being carried out on a widefield scope with a 37
>>degree incubator on it and 5% CO2 being supplied. Acquisition is
>>once every 30 minutes for 24 hours, with exposures less than 500ms
>>using a fast shutter (so fluorescent exposure is being kept to a minimum).
>>
>>
>>Thanks
>>
>>
>>Cam
>>
>>
>>
>>Cameron J. Nowell
>>Microscopy Manager
>>Centre for Advanced Microscopy
>>Ludwig Institute for Cancer Research
>>PO Box 2008
>>Royal Melbourne Hospital
>>Victoria, 3050
>>AUSTRALIA
>>
>>Office: +61 3 9341 3155
>>Mobile: +61422882700
>>Fax: +61 3 9341 3104
>><http://www.ludwig.edu.au/confocal/>Facility Website
>>
>>
>>----------
>>This communication is intended only for the named recipient and may
>>contain information that is confidential, legally privileged or
>>subject to copyright; the Ludwig Institute for Cancer Research Ltd
>>does not waive any rights if you have received this communication in error.
>>The views expressed in this communication are those of the sender
>>and do not necessarily reflect the views of the Ludwig Institute
>>for Cancer Research Ltd.
>>
>>
>>----------
>>This communication is intended only for the named recipient and may
>>contain information that is confidential, legally privileged or
>>subject to copyright; the Ludwig Institute for Cancer Research Ltd
>>does not waive any rights if you have received this communication in error.
>>The views expressed in this communication are those of the sender
>>and do not necessarily reflect the views of the Ludwig Institute
>>for Cancer Research Ltd.

Clements, Ian

Re: "Gentle" Live Cell Dye for Primary Cells

In reply to this post by Cameron Nowell

You could try something further in the red range like Calcein Red. Alternatively use a long red membrane dye like DiD (Cy5 range) or even DiR (Cy7) if you have a camera and filters for that far into the IR range (~780 nm). If you load the cells in suspension before plating they will pretty much retain the dye until they fall apart.

Any dye is going to photosensitize your cells but most of the ones in the red range will be somewhat kinder.

You didn’t mention but if you are growing your cells in any kind of matrix you could add something to the exterior environment and negatively image the cells and tubes as voids in the fluorescence background. Bob Zucker at the EPA did some work on this. If you have access to something like Imaris you can create isosurface renderings of the void volumes where the cells are.

Ian Clements

Product Manager - OMX

From: Cameron Nowell [mailto:[hidden email]]
Sent: Wednesday, May 19, 2010 3:55 PM
To: [hidden email]
Subject: "Gentle" Live Cell Dye for Primary Cells

Hi List,

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

Facility Website