oxygenate medium

[Paul Herzmark]

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Hi all,

We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen
and carbon dioxide levels to the deep parts of the tissue we bubble the
medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they
are vascularized.

Now we want to do the experiments with serum added to the medium, but the
extra protein causes the bubbling medium to foam too much.

Any suggestions on how to increase the dissolved O2 and CO2 without the
foaming?
Also, any ideas on how to monitor the resulting O2 levels?

Thanks so much.


Paul Herzmark

[hidden email]
Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]

[ricardo figueroa-2]

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Not talking from experience, but essentially bubbling is just to increase
the surface in contact with the atmosphere for gas exchange and equilibrium
to be reached. This should be possible to do by mearly having the media in
the desired atmosphere with as much surface as possible exposed. This will
probably not be as effective as bubbling but should work non the less. To
speed up things I would design a (small box or scale up as necessary)
container that is constantly supplied with the atmosphere and along the
bottom a shallow but wide laminar flow of the medium (slightly tilting the
box may be god for the flow). For monitoring the O2 levels an oxygen optode
should do the trick.

/Ricardo Figueroa

On Tue, Jun 19, 2012 at 1:08 AM, Paul Herzmark <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen
> and carbon dioxide levels to the deep parts of the tissue we bubble the
> medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they
> are vascularized.
>
> Now we want to do the experiments with serum added to the medium, but the
> extra protein causes the bubbling medium to foam too much.
>
> Any suggestions on how to increase the dissolved O2 and CO2 without the
> foaming?
> Also, any ideas on how to monitor the resulting O2 levels?
>
> Thanks so much.
>
>
> Paul Herzmark
>
> [hidden email]
> Department of Molecular and Cell Biology
> 479 Life Science Addition
> University of California, Berkeley
> Berkeley, CA  94720-3200
> [phone]
>

[Farid Jalali]

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Hi Paul, when I was still in the lab we were monitoring oxygen
concentration in media using an Oxylite Probe and Monitor like that
provided by Oxford Optronix
http://www.oxford-optronix.com/pO2monitors.htm(no commercial
interest).
We had this equipment because it is more commonly used for measuring
intra-tumoral oxygen in patient but we found it effective for diffused
Oxygen in media as well. The probes are expensive but flexible enough to
thread through a small bore perfusion inlet. It worked very well but again,
it requires an upfront investment in the equipment. Perhaps there are more
cost-effective ways of doing this, we were lucky to have the equipment
handy.
Best
Farid

On Mon, Jun 18, 2012 at 4:08 PM, Paul Herzmark <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen
> and carbon dioxide levels to the deep parts of the tissue we bubble the
> medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they
> are vascularized.
>
> Now we want to do the experiments with serum added to the medium, but the
> extra protein causes the bubbling medium to foam too much.
>
> Any suggestions on how to increase the dissolved O2 and CO2 without the
> foaming?
> Also, any ideas on how to monitor the resulting O2 levels?
>
> Thanks so much.
>
>
> Paul Herzmark
>
> [hidden email]
> Department of Molecular and Cell Biology
> 479 Life Science Addition
> University of California, Berkeley
> Berkeley, CA  94720-3200
> [phone]
>

[Deanne Veronica Catmull]

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I don't know how your experimental set up works but have you tried using a stirrer bar and a plate to slowly stir the main media reservoir whilst gassing? You could probably avoid the foaming by turning down the gas flow. It should not affect the media as the stirring would be helping to distribute the gas evenly through the media. You may also want to fit proper air filters onto the media bottle which will help regulate the pressure inside the bottle which may assist.

Regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Tuesday, 19 June 2012 9:09 AM
To: [hidden email]
Subject: oxygenate medium

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi all,

We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen
and carbon dioxide levels to the deep parts of the tissue we bubble the
medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they
are vascularized.

Now we want to do the experiments with serum added to the medium, but the
extra protein causes the bubbling medium to foam too much.

Any suggestions on how to increase the dissolved O2 and CO2 without the
foaming?
Also, any ideas on how to monitor the resulting O2 levels?

Thanks so much.


Paul Herzmark

[hidden email]
Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]

[Paul Herzmark]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thank you Deanne,
I will try those ideas. I did get a dissolved O2 meter from Milwaukee
Instruments. Unfortunately when I gas the medium with 95% O2 the level is
higher than the meter reads. Now I need additional ideas!

Paul

On Thursday, July 12, 2012, Deanne Veronica Catmull wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I don't know how your experimental set up works but have you tried using a
> stirrer bar and a plate to slowly stir the main media reservoir whilst
> gassing? You could probably avoid the foaming by turning down the gas flow.
> It should not affect the media as the stirring would be helping to
> distribute the gas evenly through the media. You may also want to fit
> proper air filters onto the media bottle which will help regulate the
> pressure inside the bottle which may assist.
>
> Regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]<javascript:;>]
> On Behalf Of Paul Herzmark
> Sent: Tuesday, 19 June 2012 9:09 AM
> To: [hidden email] <javascript:;>
> Subject: oxygenate medium
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen
> and carbon dioxide levels to the deep parts of the tissue we bubble the
> medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they
> are vascularized.
>
> Now we want to do the experiments with serum added to the medium, but the
> extra protein causes the bubbling medium to foam too much.
>
> Any suggestions on how to increase the dissolved O2 and CO2 without the
> foaming?
> Also, any ideas on how to monitor the resulting O2 levels?
>
> Thanks so much.
>
>
> Paul Herzmark
>
> [hidden email] <javascript:;>
> Department of Molecular and Cell Biology
> 479 Life Science Addition
> University of California, Berkeley
> Berkeley, CA  94720-3200
> [phone]
>

--
Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]
[phone] fax

[Thomas Horn]

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Hello Paul,
this is just an idea, haven't tried it- make an artificial gill: use a blood dialysis cartridge and make two independent fluid circuits: one closed loop in which you insert a gas-wash bottle (so it will foam in this circuit), and one that flows through the fibers into your observation chamber.
Just make sure that both liquids are identical and fresh.
Best regards,
Thomas


Dr. Thomas Horn,
The Single Cell Unit, U1.46
Department of Biosystems Science and Engineering (D-BSSE)
Swiss Federal Institute of Technology Zurich (ETH)
Mattenstrasse 26
CH 4048 Basel
Switzerland
Phone: [phone]
mail: [hidden email]



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Deanne Veronica Catmull
Sent: Freitag, 13. Juli 2012 07:46
To: [hidden email]
Subject: Re: oxygenate medium

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I don't know how your experimental set up works but have you tried using a stirrer bar and a plate to slowly stir the main media reservoir whilst gassing? You could probably avoid the foaming by turning down the gas flow. It should not affect the media as the stirring would be helping to distribute the gas evenly through the media. You may also want to fit proper air filters onto the media bottle which will help regulate the pressure inside the bottle which may assist.

Regards,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Tuesday, 19 June 2012 9:09 AM
To: [hidden email]
Subject: oxygenate medium

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi all,

We do 2P microscopy of thick tissue explants. To deliver appropriate oxygen and carbon dioxide levels to the deep parts of the tissue we bubble the medium with 95% O2 and 5% CO2. That keeps the cells as happy as when they are vascularized.

Now we want to do the experiments with serum added to the medium, but the extra protein causes the bubbling medium to foam too much.

Any suggestions on how to increase the dissolved O2 and CO2 without the foaming?
Also, any ideas on how to monitor the resulting O2 levels?

Thanks so much.


Paul Herzmark

[hidden email]
Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]

[Paul Herzmark]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Thomas,
Thank you for that idea. I will look into it and report back!

Paul

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]
[phone] fax


On Tue, Jul 17, 2012 at 12:32 AM, Horn Thomas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Paul,
> this is just an idea, haven't tried it- make an artificial gill: use a
> blood dialysis cartridge and make two independent fluid circuits: one
> closed loop in which you insert a gas-wash bottle (so it will foam in this
> circuit), and one that flows through the fibers into your observation
> chamber.
> Just make sure that both liquids are identical and fresh.
> Best regards,
> Thomas
>
>
> Dr. Thomas Horn,
> The Single Cell Unit, U1.46
> Department of Biosystems Science and Engineering (D-BSSE)
> Swiss Federal Institute of Technology Zurich (ETH)
> Mattenstrasse 26
> CH 4048 Basel
> Switzerland
> Phone: [phone]
> mail: [hidden email]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Deanne Veronica Catmull
> Sent: Freitag, 13. Juli 2012 07:46
> To: [hidden email]
> Subject: Re: oxygenate medium
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I don't know how your experimental set up works but have you tried using a
> stirrer bar and a plate to slowly stir the main media reservoir whilst
> gassing? You could probably avoid the foaming by turning down the gas flow.
> It should not affect the media as the stirring would be helping to
> distribute the gas evenly through the media. You may also want to fit
> proper air filters onto the media bottle which will help regulate the
> pressure inside the bottle which may assist.
>
> Regards,
> Deanne.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Paul Herzmark
> Sent: Tuesday, 19 June 2012 9:09 AM
> To: [hidden email]
> Subject: oxygenate medium
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> We do 2P microscopy of thick tissue explants. To deliver appropriate
> oxygen and carbon dioxide levels to the deep parts of the tissue we bubble
> the medium with 95% O2 and 5% CO2. That keeps the cells as happy as when
> they are vascularized.
>
> Now we want to do the experiments with serum added to the medium, but the
> extra protein causes the bubbling medium to foam too much.
>
> Any suggestions on how to increase the dissolved O2 and CO2 without the
> foaming?
> Also, any ideas on how to monitor the resulting O2 levels?
>
> Thanks so much.
>
>
> Paul Herzmark
>
> [hidden email]
> Department of Molecular and Cell Biology
> 479 Life Science Addition
> University of California, Berkeley
> Berkeley, CA  94720-3200
> [phone]
>