fluorescent medium
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Hi all,
Does anyone have any suggestions for low fluorescing mammalian cell media? We use DMEM and RPMI media without phenol red and they both have too much background fluorescence. The fluorescence is strong in the GFP channel with both one photon and two photon (around 900) excitation. If you have any idea what is fluorescing in those media please tell me. I know I will eventually have to test variations to find what also keeps the cells happy. Thanks for any and all ideas! Paul Paul Herzmark Specialist [hidden email] Department of Molecular and Cell Biology 479 Life Science Addition University of California, Berkeley Berkeley, CA 94720-3200 (510) 643-9603 |
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Hi Paul:
My guess would be riboflavin (emission peak ~540 nm).
According to my notes, DMEM contains 0.4 mg/L riboflavin. One possible
alternative is Ham's F12, which is only 0.04 mg/L
riboflavin.
Iain
Hi all,From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark Sent: Monday, December 22, 2008 2:39 PM To: [hidden email] Subject: fluorescent medium Does anyone have any suggestions for low fluorescing mammalian cell media? We use DMEM and RPMI media without phenol red and they both have too much background fluorescence. The fluorescence is strong in the GFP channel with both one photon and two photon (around 900) excitation. If you have any idea what is fluorescing in those media please tell me. I know I will eventually have to test variations to find what also keeps the cells happy. Thanks for any and all ideas! Paul Paul Herzmark Specialist [hidden email] Department of Molecular and Cell Biology 479 Life Science Addition University of California, Berkeley Berkeley, CA 94720-3200 (510) 643-9603 |
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In reply to this post by Paul Herzmark
I can tell you from personal experience formulating low fluorescence
yeast media that riboflavin and folic acid are both fluorescent. I also found that commercial media without these vitamins was still fluorescent, but that if I made it myself from components it was completely non-fluorescent. So I suspect that some of the commercial (yeast) media are not that pure and contain fluorescent impurities. No idea how much of this will apply to mammalian cell media, but I would definitely try removing riboflavin and folic acid if present. Riboflavin was the more fluorescent of the two. Kurt Paul Herzmark wrote: > Hi all, > Does anyone have any suggestions for low fluorescing mammalian cell media? > > We use DMEM and RPMI media without phenol red and they both have too > much background fluorescence. The fluorescence is strong in the GFP > channel with both one photon and two photon (around 900) excitation. > > If you have any idea what is fluorescing in those media please tell > me. I know I will eventually have to test variations to find what also > keeps the cells happy. > > Thanks for any and all ideas! > Paul > > > Paul Herzmark > Specialist > [hidden email] <mailto:[hidden email]> > > Department of Molecular and Cell Biology > 479 Life Science Addition > University of California, Berkeley > Berkeley, CA 94720-3200 > (510) 643-9603 > -- Kurt Thorn, PhD Director, Nikon Imaging Center University of California San Francisco UCSF MC 2140 Genentech Hall Room S252 600 16th St. San Francisco, CA 94158-2517 http://nic.ucsf.edu phone 415.514.9709 fax 415.514.4300 |
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In reply to this post by Johnson, Iain
Hi there I agree that riboflavin is the likely
cause of your autofluorescence, more so than phenol red. F12 has less
riboflavin but a lot of Vit B12 which also fluoresces in that range if I
remember well. MEM has no Vit B12 and less riboflavin as DMEM so it could be
worth trying it along with F12. It is also important to use medium that is
in as fresh as possible so exposed to light as little as possible and with
little temperature variation during storage. Warming up the whole bottle of
medium at every passage is fine if the bottle is used within a week or 10 days but
otherwise I would warm up aliquots and keep the stock in the fridge. Happy New Year! Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 +46 (0)8 608 9240 From:
Hi Paul: My guess would be riboflavin (emission
peak ~540 nm). According to my notes, DMEM contains 0.4 mg/L
riboflavin. One possible alternative is Ham's F12, which is
only 0.04 mg/L riboflavin. Iain From: Hi all, |
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Hi, For conventional fluorescence imaging, Chroma have a GFP filter
set modified to minimise detection of flavin autofluorescence: http://www.chroma.com/index.php?option=com_products&Itemid=53&task=details&productType=set&id=116 Anybody know the excitation spectra of riboflavin? Chroma also
have a GFP UV filter set that excites at 375-425, making use of GFP UV’s 395 nm
excitation peak rather than its less optimum 478 peak: http://www.chroma.com/index.php?option=com_products&Itemid=53&task=details&productType=set&id=70 No commercial interest. Daniel ------------------------------------------------ Daniel Brotchie Ophthalmology School of Clinical Sciences University Clinical Departments The Duncan Building Daulby Street LIVERPOOL L69 3GA UK Tel: +44 (0) 151 706 4017 Fax: +44 (0) 151 706 5934 E-mail [hidden email] From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Sylvie Le Guyader Hi there I agree that riboflavin is the likely cause of your
autofluorescence, more so than phenol red. F12 has less riboflavin but a lot of
Vit B12 which also fluoresces in that range if I remember well. MEM has no Vit
B12 and less riboflavin as DMEM so it could be worth trying it along with F12. It is also important to use medium that is in as fresh as possible
so exposed to light as little as possible and with little temperature variation
during storage. Warming up the whole bottle of medium at every passage is fine
if the bottle is used within a week or 10 days but otherwise I would warm up
aliquots and keep the stock in the fridge. Happy New Year! Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 Huddinge Sweden +46 (0)8 608 9240 From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Johnson, Iain Hi Paul: My guess would be riboflavin (emission peak ~540 nm).
According to my notes, DMEM contains 0.4 mg/L riboflavin. One possible
alternative is Ham's F12, which is only 0.04 mg/L riboflavin. Iain From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Paul Herzmark Hi all, |
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Hi,
The company, Brain Bits, makes a product
Hibernate A 500 low fluorescence . If you search their site you will find a graph
that shows that phenol red acts as a dye quencher rather than as an
autofluorescent source itself. Quenches the flavins and folic acid and
other dyes of course
So when you remove the phenol red, you need to lower
the flavin/folic acid concentrations, which they have done. So really
not just low fluorescence media, but this data would suggest an additional
benefit as a fluorescence brightener if phenol red
is removed. Anyone compare?
No commercial interest.
Mike Ignatius
Molecular Probes/Life Technologies From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Brotchie, Daniel Sent: Wednesday, January 07, 2009 8:50 AM To: [hidden email] Subject: Re: fluorescent medium Hi, For
conventional fluorescence imaging, Chroma have a GFP filter set modified to
minimise detection of flavin autofluorescence: http://www.chroma.com/index.php?option=com_products&Itemid=53&task=details&productType=set&id=116 Anybody
know the excitation spectra of riboflavin? Chroma also have a GFP UV filter set
that excites at 375-425, making use of GFP UVs 395 nm excitation peak rather
than its less optimum 478 peak: http://www.chroma.com/index.php?option=com_products&Itemid=53&task=details&productType=set&id=70 No
commercial interest. Daniel ------------------------------------------------ Daniel
Brotchie Ophthalmology School
of Clinical Sciences University
Clinical Departments The
Duncan Building Daulby
Street LIVERPOOL L69
3GA UK Tel:
+44 (0) 151 706 4017 Fax:
+44 (0) 151 706 5934 E-mail
[hidden email] From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of
Sylvie Le Guyader Hi
there I agree
that riboflavin is the likely cause of your autofluorescence, more so than
phenol red. F12 has less riboflavin but a lot of Vit B12 which also fluoresces
in that range if I remember well. MEM has no Vit B12 and less riboflavin as DMEM
so it could be worth trying it along with F12. It is
also important to use medium that is in as fresh as possible so exposed to light
as little as possible and with little temperature variation during storage.
Warming up the whole bottle of medium at every passage is fine if the bottle is
used within a week or 10 days but otherwise I would warm up aliquots and keep
the stock in the fridge. Happy
New Year! Med
vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie
Le Guyader Dept of
Biosciences and Nutrition Karolinska
Institutet Novum 14157
Huddinge Sweden +46 (0)8
608 9240 From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of
Johnson, Iain Hi
Paul: My guess
would be riboflavin (emission peak ~540 nm). According to my notes, DMEM
contains 0.4 mg/L riboflavin. One possible alternative is Ham's F12,
which is only 0.04 mg/L riboflavin. Iain
From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Paul
Herzmark Hi all, |
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