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Dear Group Members,
I am trying to optimize MDCK permeability assay on 6 well PET
inserts, 3 um. I get TEER values to be above 200 after 4 days but
lucifer yellow test done at 100 uM with 15, 30, 45, 60, 90 and 120
min time points, shows higher permeability values (analysis by
fluorimetry). Literature shows ideally it should be below 30 nm/sec.
Also I see that the cumulative amount transported saturates at higher
time points.
Is there any substitute for LY other than mannitol and PEG?
How much important is LY if TEER values are ok?
Thanks in advance!
Regards
Tripta Bansal
PhD Scholar
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The following message was posted to: PharmPK
Dear Tripta,
The LY criterion you mention looks reasonable for the Caco-2 cell model,
but it may depend on the cell model (any opinion in the Group about
that?).
It is not clear to me whether you determined TEER only before
experiment, or before and after. The former situation tells you that you
may run a transport experiment on that seeded filter. The latter, as you
may know, is used to check the integrity of cell monolayer over the
whole experiment, in combination with the permeability of an internal
(co-incubated) standard such as radiolabeled mannitol or LY.
Have you tried to correlate TEER decrease, LY permeability, and
permeability of another co-incubated low permeability compound
(acyclovir ? atenolol ?).
Best regards,
Frederic MASSIERE, PhD
ADME-PK Group Director, Oroxcell
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Dear all,
I run hmdr1-MDCK permeability assay using only LY to assess the
monolayer integrity.
I have been using the same 30 nm/sec cut-off for years and I have
found it reasonable considering it "jumps" to a very high level when
the monolayer integrity is compromised.
Tripta,
I would suggest also considering mycoplasma contamination which as I
am sure you well know, would affect your cell cultures, by damaging
the monolayer integrity and thus increasing LY values. Considering
mycoplasma contamination can be difficult to confirm it may just be
easier to defrost another batch of cells and try with that one.
Hope this help.
Regards,
Monica.
Monica Rigo
Bioanalytics, Metabolism and in vitro Technologies
GlaxoSmithKline
Via Fleming 4, 37135 Verona- ITALY
Psychiatric CEDD DMPK Verona ITALY
Tel. +39 045 821 8770
Fax. +39 045 821 8153
E-mail. monica.e.rigo.-a-.gsk.com
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The following message was posted to: PharmPK
>>>I would suggest also considering mycoplasma contamination which
as I am sure you well know, would affect your cell cultures, by
damaging the monolayer integrity and thus increasing LY values.
Considering mycoplasma contamination can be difficult to confirm it
may just be easier to defrost another batch of cells and try with
that one.
Is there a way to confirm mycoplasma contamination ? If you have
frozen down vials with contaminated cells you would not see any change.
--
Nagdeep Giri,
Graduate student
Dept. of Pharmaceutics, University of Minnesota
308 Harvard Street SE,3-140 WDH, Minneapolis, MN 55455
(612) 625-2446/626-3809 ; E-mail: giri0003.-at-.umn.edu
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The following message was posted to: PharmPK
All,
I would suggest that if you think mycoplasma is a potential issue, you
look into using Plasmocin(TM) as it will eliminate this problem. This
compound is very specific for only mycoplasma and reportedly works well.
We use it in a prophylactic manner and have had no issue with it
changing any of our cultures.
Cheers-
Russell
--
VaxDesign Corporation
Russell G Higbee, MS, PhD, DVM
Senior Scientist
rhigbee.-a-.vaxdesign.com
2721 Discovery Dr.
Suite 400
Orlando, FL 32826
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Dear all,
Recent reports estimate Mycoplasma contamination in up to 63% of all
cell cultures.
Mycoplasma cannot be detected by visual inspection and may not
noticeably affect cell culture growth rates.
However, Mycoplasma infection has been shown to alter DNA, RNA and
protein synthesis, induce chromosomal aberrations and cause
alterations or modifications of host cell plasma membrane antigens.
Dear Nagdeep,
I'm sure there are some kits to determine it, but I have never used
them as I found them too expensive in terms of money and time.
In the past, I have been "lucky" only changing the batch line and I
found all the parameters reasonable. I wish Tripta the same good luck
I had!
Regards,
Monica Rigo
Bioanalytics, Metabolism and in vitro Technologies
GlaxoSmithKline
Via Fleming 4, 37135 Verona- ITALY
Psychiatric CEDD DMPK Verona ITALY
Tel. +39 045 821 8770
Fax. +39 045 821 8153
E-mail. monica.e.rigo.-at-.gsk.com
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The following message was posted to: PharmPK
Dear All,
We have used such kits to get rid off Mycoplasma infection from cell
lines received from academia with great success!
Please write to me directly and I will be able to provide you with
the details of the kit.
Best Regards,
Gabor
Gabor Heltovics, M.Sc, MBA
Business Development Director
SOLVO Biotechnology
Central Hungarian Innovations Center
H-2040 Gyar u. 2.
Budaoers, Hungary
Tel: +36-23-503-937
Fax:+36-23-503-941
www.solvo.com
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Dear Giri,
I have already used 2 kits to determine Mycopalsma infection in our
different cell lines : MycoAlert Mycoplasma Detection Kit from
Cambrex (ref LT07-118) and Mycoplasma Detection Kit from Roche (ref 1
296 744).
The kit provided by Cambrex is quicker (+/- 1 hour) but the result is
not always clearcut. The test provided by Roche is an Elisa test
which is more specific and sensitive but takes ca 24h.
Regards.
Brigitte GERIN
Research Scientist
ADME and Interactions, UCB
Chemin du Foriest
1420 Braine l'Alleud
BELGIUM
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Dear All,
In continuation with discussion on MDCK Permeability assays, which is
the best suitable MDCK strain for doing drug permeability assays -
Strain I or Strain II. As per literature, Strain I show high TEER
values where as Strain II shows less. Can anybody suggest the
commercial source from which MDCK strain I can be obtained. We are
using Caco2 filter (96 well) to grow MDCK cells (purchased from ATCC,
Catalog No. CCL- 34). We have tried different seeding densities, but
our TEER values are almost similar to blank wells. Can anyone suggest
the reasons for poor TEER values. The cells look fine when grown in
TC flasks or in TC plates. We are using DMEM media with 10% FBS
between Passage No. 64-66.
Thanks in advance,
SB
Ph.D Scholar
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The following message was posted to: PharmPK
Hi,
We successful execute the MDCK assay on a weekly basis using the Corning
Costar (Cat # 3397 or equivalent) 24 well with 0.4um pore cluster insert
And the Corning Costar (Cat # 3524 or equivalent) 24 well with lid. Our
TEER measurements are very consistent.
Cells are plated at a density of 0.5x10^6 cells/mL in 75uL and allowed
to grow for 4 days with media change on day 2. The cells were from ATCC
as well.
Hope this helps!
Good luck!
Lory
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