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MDR1-MDCK - Setting up assay (Mar/27/2010 )

Hi all,

I'm trying to setup MDR1-MDCK assay. I have lots of literature but most seem to be lacking a detailed procedure.

I know most start at 10um test compound. However what is volume of buffer in the 24 well plates?

Also where can I purchase MDR1-MDCK cell line? Any help would be greatly appreciated!

Thank you

-firstlast2929-

10 uM is a concentration, it is independent of volume. I use 0.5 ml medium per well of a 24 well plate, but you could probably range between 0.15 and 1.0 ml without too many side effects.

-bob1-

bob1 on Mar 28 2010, 08:07 PM said:

10 uM is a concentration, it is independent of volume. I use 0.5 ml medium per well of a 24 well plate, but you could probably range between 0.15 and 1.0 ml without too many side effects.



Thanks for the reply!

Do you know where I can purchase the cell line?

Thanks

-firstlast2929-

firstlast2929 on Mar 28 2010, 06:07 PM said:

bob1 on Mar 28 2010, 08:07 PM said:

10 uM is a concentration, it is independent of volume. I use 0.5 ml medium per well of a 24 well plate, but you could probably range between 0.15 and 1.0 ml without too many side effects.



Thanks for the reply!

Do you know where I can purchase the cell line?

Thanks



ATCC, or you can ask someone to give you one vial.

-Eric-

Eric on Mar 31 2010, 12:44 AM said:

firstlast2929 on Mar 28 2010, 06:07 PM said:

bob1 on Mar 28 2010, 08:07 PM said:

10 uM is a concentration, it is independent of volume. I use 0.5 ml medium per well of a 24 well plate, but you could probably range between 0.15 and 1.0 ml without too many side effects.



Thanks for the reply!

Do you know where I can purchase the cell line?

Thanks



ATCC, or you can ask someone to give you one vial.


Sry, no MDR1-MDCK cells in ATCC it seems.

-Eric-

Thanks for all the help!

It seems most people are getting the cells from NIH. Does anybody have a contact there I can contact about getting a vial?

Also I am little confused about something.

I will be using LC/MS for quantifying the data.

If 10um is added to the A-side, after 1 hour incubation, I will sample from A and B sides. How do I quantify? Do I need use a reference at 10um? Or do I just sample from A and B compare the the mass area from the two?

Thanks again for any help!

-firstlast2929-

The reason MDR-1 MDCK cells don't appear to in the regular ATCC catalog is because they are covered by two US patents, and if you need to start using them, there's an annual fee you'll need to pay, in addition to a lofty charge per vial of actual cells. You need to talk to the Office of Technology Transfer at the NIH about procuring a use license for these cells, and paying the licensing fee. They manage the patented materials, and once you have a license with them, then the ATCC will release a vial of cells to you or your company. The person I was put in contact with about obtaining a license for these cells is:

David Lambertson, PhD
Senior Licensing and Patenting Manager
301-435-4632
lambertsond@od.nih.gov

I got his contact information from the NIH OTT website.

As for the assay, 10ÁM is pretty high. I only say that because it's possible you might start to saturate the MDR-1 transporter if you're dealing with a potent substrate. What you want to see is high basolateral-to-apical flux but much reduced apical-to-basolateral flux with these cells for any compound that's a substrate of MDR-1. Common positive controls include Vinblastine, Verapamil, and Cyclosporin-A. Digoxin also works but doesn't fly very well with MS based detection. Propranolol is a good negative control as it has very high permeability in both directions, and hence no efflux ratio (B-to-A transport / A-to-B transport).

1ÁM to 2ÁM is a good place to start. This conc should give you really good MS detection, even for poorly fluxed compounds where there's only a little compound in the receiver side. We use an API3000 MS, and it works just fine. For quantitation, what we do is monitor the molar flux of compound in time, say a 1 hour incubation, and set it up as a ratio against the molar mass of compound you started with on the donor side. Your calculation must take into account the receiver side volume because concentration is dependent on it. Equal flux into a smaller volume will yield higher concentration (hence, higher peak areas). Hope this helps.

-KDeMent-