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The following message was posted to: PharmPK
Hello Everybody,
I need some advice concerning Caco-2 experiments. We use a Caco-2 kit
from
BD (3-day model) to profile compounds for absorption characteristics.
To check the quality of the cell layer we use TEER measurements (all
wells)
before we load the wells with test compounds, we have one well reserved
for
lucifer yellow as layer integrity test and after the permeability
experiment we check all layers with lucifer yello again. So far so good.
However, I have the impression, that the well-to-well reproducibility is
rather high (0.8 to 1.3 E-6 cm/sec for lucifer yellow). From literature
I
know that the 3-day model is much "leakier" than the other models.
Nevertheless, we want to keep up with this model, since it requires much
less time resources.
Now my question: Is it advisable to incubate the unknown compounds
together
with lucifer yellow as internal reference standard
(2-in-one-experiment) or
is this a bad idea? Does anybody uses internal reference standards, and
if
yes, what compounds do you use?
(We cannot assess radioactive labelled componds).
All suggestions are wellcome!
Thank you very much in advance!
kind regards,
markus
markus rueckert
morphochem AG
gmunder str. 37-37a
81379 muenchen
tel. ++49-89-78005-0
fax ++49-89-78005-555
http://www.morphochem.de
markus.rueckert.-at-.morphochem.de
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Dr. Rueckert,
Although we have not used the 3-day model, when we run the 21-day Caco
analyses we routinely mix the test compound with lucifer yellow as an
internal control. This has proven to be beneficial several times in
determining if the permeability data should be trusted and if a
compound is potentially toxic to the cells thereby giving a false high
permeability. The addition of lucifier yellow to control compounds
does not seem to affect the overall interpretation of their cell
permeability.
I hope this helps.
Mark Schurdak
Group Leader
Biological Screening
Abbott Laboratories
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