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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
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---------------
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)
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-----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
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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
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