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> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
We found years ago when doing studies of adhesion of cells to
protein-coated substrates that HEPES added at the manufacturer's
recommended concentration was not totally toxic, but did produce anamalous
spreading patterns on the control serum-coated dishes/coverslips. With
5-10mm HEPES, sreading was normal. In addition to retaining some
bicarbonate in the medium, low levels of phosphate are also required.
As for the mentioned phototoxicity, remember that phenol red can be
phototoxic.
Colin Izzard
>
> We have found that HEPES may alter the ability of some crawling mammalian
> cell types to change polarity in response to chemoattractant. On the
> other
> hand, a majority of our live cell experiments are done with HEPES buffer
> because when we deal with new cell types, usually we take a baby step back
> to characterize their behavior in buffered vs. unbuffered media. Do you
> really want to come to conclusions about protein translocation in response
> to stimuli or cell directional crawling only to find out that it's an
> artefact of pH stabilization?
> -Michael Cammer
>
> At 03:39 PM 03/14/05 +0100, you wrote:
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Ian -
>>
>>can you give me a reference of a study that disproves the findings that
>>HEPES enhances toxicity of light-exposed culture media (it wasn't claimed
>>that HEPES itself is toxic)?
>>
>>Thanks,
>>
>>Beat
>>
>>At 23:15 13-03-2005, you wrote:
>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Hello Andy
>>>
>>>I'm sure the list must be tired of me saying this, but why do you need
>>>to use CO2 buffering anyway? It is much better to use a HEPES buffered
>>>salt solution / medium. Old notions that HEPES is toxic were disproven
>>>years ago (you do need to keep some bicarbonate in the mix...) . It is
>>>almost impossible to get adequate pH control using a CO2/Bicarbonate
>>>buffering in a small volume, with or without a flow-through... it's
>>>just too dependent on flow rate / temperature / bubbling rate / size
>>>and shape of the chamber etc...
>>>
>>>There are plenty of recipes around, but if you want ours, I can send it
>>>to you.
>>>
>>>cheers
>>>
>>>IAN
>>>
>>>On Saturday, March 12, 2005, at 05:46 AM, Andrew Resnick wrote:
>>>
>>>>Search the CONFOCAL archive at
>>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>>Several of us are putting in a NCRR Shared Instrumentation Grant for an
>>>>upright scope to eprform live-cell studies. There does not appear to
>>>>be a
>>>>commercial solution for atmospheric or pH control- those systems are
>>>>made
>>>>for inverted scopes. We have heard that gently superfusing a 5% CO2
>>>>mixture over the top of the cells is sufficient due to the density of
>>>>CO2
>>>>with respect to air. Is there someone who has done this? If so,
>>>>please
>>>>let me know as I would like to include a reference to this method in
>>>>the
>>>>application. Thanks in advance,
>>>>
>>>>Andy
>>>>
>>>>Andrew Resnick, Ph. D.
>>>>Fellow
>>>>Department of Physiology and Biophysics
>>>>Case Western Reserve University
>>>>216-368-6899 (V)
>>>>216-368-4223 (F)
>>>
>>>* * * * * * * * * * *
>>>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
>
> ____________________________________________________________________________
> Michael Cammer Analytical Imaging Facility Albert Einstein Coll. of
> Med.
> Jack & Pearl Resnick Campus 1300 Morris Park Ave. Bronx, NY
> 10461
> (718) 430-2890 Fax: 430-8996 URL:
> http://www.aecom.yu.edu/aif/
> **This electronic transmission contains information that is
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>
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