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Hello, Ian
Thank you for your reply. HEPES buffer is certainly an option but I
will have to check whether sensitive cells - such as EPC's - will
tolerate a different type of buffering system.
Thank you
Judy
>>> [log in to unmask] 6/17/2004 7:22:21 PM >>>
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Hello Sarah and Judy
Although I haven't done quite this sort of experiment, I do do a lot
of
in vitro recording of various things and occasionally image what is
going on. In our experience over more than 25 years, you will never
solve the CO2 control problem in a reliable way - it is just too
dependent on temperature, gas flow rates, solution flow rates, gas vs
liquid volume and who knows what else. About 20 years ago we switched
to HEPES buffered solutions, gassed with an oxygen mixture, and pH
control just isn't a problem any more (at least not because of gas
mixtures...). There is a school of thought out there that HEPES is
toxic to cells, but this isn't really true. The problem was that some
HEPES buffered media dropped out the bicarbonate, since it was thought
that you don't need it for the buffering any more. Well, you don't in
a
chemical sense, but most cell types have a requirement for some
bicarbonate in the extracellular fluid, so we always include it in any
HEPES buffered medium. Indeed, most commercial culture media that are
buffered with HEPES do include the bicarb...
I'm not sure if that really is the issue you had, but I hope that
helps. I can send you a recipe for a pretty standard HEPES buffered
salt solution, if you like (or just look up any of our recent papers -
we always include it...)
IAN
On Friday, June 18, 2004, at 02:51 AM, Locknar, Sarah A wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi-
> It sounds like you're trying to regulate the CO2/ humidity in the
> entire
> incubator? If that's what you're trying do do, it probably won't
work.
> We do the following:
> 1. Let temp in chamber stabilize a MINIMUM of 3 hours (overnight is
> better)
> 2. puncture the side of the plastic dish with a heated needle (we
go
> through the top AND bottom at once)
> 3. insert a new needle. This needle is connected directly to the
> output of CO2 bubbling through water (thus the CO2 is hydrated) We
use
> the Solent chamber and CO2 enrichment controller but I'm not
convinced
> it's completely necessary, provided you have a low-flow regulator on
> the
> gas tank. We use a tank that's 5% CO2, 20% O2, 75% N2. The dish
> shouldn't need an outlet needle since they are not airtight-the gas
> tends to leak out.
>
> This setup has worked with experiments up to 18 h long for us.
> Feel free to give me a call if this isn't clear.
> Good luck-
> Sarah
>
-----------------------------------------------------------------------
> -
> ---------------
> Sarah Locknar, Ph.D.
> Director, Neuroscience COBRE Imaging / Physiology Core
> College of Medicine, University of Vermont
> E015 Given Building
> 89 Beaumont Ave.
> Burlington, VT 05405
> 802-656-0413
> 802-656-8704 (fax)
>
-----------------------------------------------------------------------
> -
> ---------------
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
> Behalf Of Judy Trogadis
> Sent: Thursday, June 17, 2004 12:53 PM
> To: [log in to unmask]
> Subject: live cell website
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear confocalists
>
> We have started long-term live cell experiments (>24 hours) and
> although
> I have followed basic instructions learned over the years, there are
> still problems with our setup.
>
> If anyone is interested in the details, we have a fair-sized
> plexiglass
> incubator housing the microscope stage into which I have put a
> water-filled flask through which CO2 is bubbled, we have a heater
> stage, a blower providing warm air and an extra container of water
for
> humidity. Still, after several hours, when the temperature should
have
> stabilized, we see drift in focus, condensation on the inside of the
> lid
> of the cell culture dish and inadequate humidity. CO2 is not
regulated
> accurately.
>
> Does anyone know of any webiste or other literature with lots of
> pictures to see how others have designed and used live cell setups.
>
> Thank you in advance
> Judy
>
> Judy Trogadis
> Bio-Imaging Coordinator
> St. Michael's Hospital, 7Queen
> 30 Bond St.
> Toronto, ON M5B 1W8
> Canada
> ph: 416-864-6060 x6337
> pager: 416-685-9219
> fax: 416-864-6043
> [log in to unmask]
>
>
* * * * * * * * * * *
Prof Ian Gibbins
Anatomy & Histology
Flinders University
GPO Box 2100
Adelaide SA 5001
AUSTRALIA
[log in to unmask]
voice: +61-8-8204 5271
fax: +61-8-8277 0085
http://som.flinders.edu.au/FUSA/Anatomy/
http://www.flinders.edu.au/neuroscience
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