Dear Karatza: As Richard mentions, the functional assay for detection of Pgp using calcein AM (in an accumulation assay) is a good test for funtion. Other dyes, the most common being Rhodamine 123 (which is what we use), also work well in a retention assay. For antigen detection, the mAb MRK-16 is one of the best for detecting lower levels of the protein (i.e.: not the logarithmically higher levels commonly seen on a cell line); you can check the archive of this list, but people have complained about some directly conjugated anti-Pgp mAbs not detecting the protein at low [yet clinically relevant] levels of expression, and we have found this also to be the case (Leukemia Research, 25:1127-1135). For MRP1 detection, Calcein AM in a retention assay works well - BCECF-AM is not specific for the MRP-1 transporter (Cytometry, 46:105-113), whereas the two antibodies most commonly used for detecting the MRP-1 protein are MRPm6 (mouse monoclonal) and MRPr1 (rat monoclonal). An relaible functional assay does not exist for LRP, and the antibody for it that is the most common one is LRP-56. In terms of the other MDR proteins - MDR2 through MDR6 and others, such as BCRP-1, the verdict still seems to be out on what is the "best/optimal" way to detect these by antigen expression or function by flow. BCRP-1 has a functional assay (people have used Bodipy as well as the actual drug Mitoxantrone, both of whihc work nicely actually), but the key here is the reversal aganet - fumitremorgin C - which is not to be commercially found these days (or at least has evaded the radar in my searches in the past few months). Best of luck! do ----------------------------------------------------------- Douglas P. Olson Experimental Hematology/AIDS Research Center Massachusetts General Hospital Harvard Medical School 149 13th Street Boston, MA 02129 (617)724-2668 - Phone (617)726-4691 - Fax Dolson4@partners.org - Email > -----Original Message----- > From: Richard Haugland [SMTP:richard.haugland@probes.com] > Sent: Thursday, April 11, 2002 11:52 AM > To: Cytometry Mailing List > Subject: Re: mdr and flow > > > Possibly the easiest assay for P glycoprotein-mediated MDR uses calcein > AM, which is selectively excreted by MDR cells rather than an antibody. > > http://www.probes.com/handbook/sections/1505.html > > Biochim Biophys Acta 1994 May 11;1191(2):384-8 > > Calcein accumulation as a fluorometric functional > assay of the multidrug transporter. > > Hollo Z, Homolya L, Davis CW, Sarkadi B. > > National Institute of Haematology, Blood > Transfusion and Immunology, Budapest, Hungary. > > Acetoxymethyl ester (AM) derivatives of various > fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are > actively extruded > by the multidrug transporter (MDR1, > P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, > 21493-21496). In the present > paper we show that the measurement of the > accumulation of a fluorescent cell viability marker, calcein, can be > effectively used as a > rapid and sensitive fluorometric and flow > cytometric assay for studying P-glycoprotein function. The rate of > calcein accumulation in > human MDR1-expressing cells is significantly > lower than in the control cells, while various drug-resistance reversing > agents (verapamil, > vinblastine, oligomycin, cyclosporin A and UIC2 > monoclonal antibody) greatly increase calcein trapping only in the > MDR1-expressing > cells. Since calcein-AM is not fluorescent and > free calcein is not a substrate of the multidrug transporter, the assay > is readily applicable > for rapid kinetic studies of the MDR1 function. > Calcein has a high fluorescence intensity in the visible range, thus > changes in calcein > uptake can be easily visualised and > MDR1-expressing and control cells separated by conventional flow > cytometry. > > > > Karatza wrote: > > > Dear all,Does anybody has any experience in mdr and flow ? What are > > the best antibodies?
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