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Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye
J E Dinchuk1, K A Kelley, G N Callahan
1Institute for Molecular Biologicals, Miles Research Center, West Haven, CT 06516.
Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.
Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.