Bryone, CFDA appears, according to Molecular Probes to be pumped out faster than other dyes (calcein) used for MRP assessment. I am assuming you are loading with the acetoxymethyl ester (5-CFDA-AM). If you are not, the anion will not get into the cell. If you are loading with the succinic ester, that is a tracking dye that binds to cell proteins and tracks cell division. I would use calcein AM, and that loads well with HBSS. In the ester form, however, these probes are pumped out by MDR at the membrane before they actually enter the cytoplasm and are broken down by esterases. If your cells have variable expression of MDR, that may account for variablity in loading. In that case, load in the presence of verapamil, 10 ug/ml, then wash out the verapamil. MD571 is, at higher concentrations necessary than LTC4 receptor inhibition, an MRP1 inhibitor. ron on 4/23/02 3:48 AM, Bryone Kuss at bryone.kuss@flinders.edu.au wrote: > Dear All, > > We have been having problems with the transport of CFDA in functional assays > of MRP1. > > PBS+gluc is the recommended solution for the assay but our cells do not like > it and die rather quickly. The effects of using Hanks in place of this were to > significantly reduce the transport of CFDA out of the cells, although the > cells were much healthier. Alternatively there is a creatine and Tris buffered > PBS solution but we have not tried this. > What do others find? > > Secondly, we have found that MK571 works unreliably as an inhibitor of MRP1 in > media + FCS. Does anyone have any experience they would be willing to share > about either of these agents? > > Many thanks, Bryone > > Bryone J Kuss > > MBBS, PhD > Department of Haematology and Genetic Pathology > FMC, Flinders University > South Australia 5042 > > Fax 618 8204 5450 > Ronald L. Rabin, M.D. Senior Staff Fellow Laboratory of Immunobiochemistry DBPAP/OVRR Center for Biologics Evaluation and Research U.S. Food and Drug Administration 29 Lincoln Drive (MSC-4555) Building 29, Room 129 Bethesda, MD 20892-4555 phone: 301.496.8806 fax: 301.402.5177 email: rr84g@nih.gov
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