Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. acid per ml. Ascorbic acid was used as an antiphotooxidation agent. RNF49 The cells were imaged with an inverted confocal laser scanning microscope as described previously (23). Relative fluorescence was measured in cells by focusing on areas of cytoplasm that were away from the nucleus and free of vacuoles or areas of sequestered calcein. The calcein extrusion by E6 cells was variable, presumably reflecting different degrees of plasma membrane Pgp expression. As a result, the calcein fluorescence of E6 cell cultures varied between cultures and between passages, depending on which clonal expansions of cells dominated the culture. To reduce the variance in relative fluorescence between treatment groups in an experiment, all cells used in a given experiment came from the same culture and passage number. Similarly, while the laser and gain settings were optimized for each experiment, they were m-Tyramine hydrobromide kept constant throughout a given experiment. In one experiment designed to determine the effect of Pgp and MRP inhibitors on calcein fluorescence, uninfected cells were plated as m-Tyramine hydrobromide above and exposed to 10 M verapamil, 10 m-Tyramine hydrobromide M cyclosporin A, or 100 M probenecid. The carrier for cyclosporin A was ethanol (final concentration, 0.1%). After 45 min in medium containing the transporter inhibitor, calcein AM was added to the medium for an additional 15 min as described above. Ethanol and DMSO carrier controls were carried out as appropriate. This carrier did not affect cell fluorescence at the concentrations used. In order to determine if there was a calcein AM or calcein extrusion pump in the parasite, heavily infected cells were broken up by passing a cell suspension through a 26-gauge needle three times. The most abundant parasite stage, the mature spore, did not load with calcein, presumably due to its complex spore coat. Meronts and other single parasite stages were difficult to distinguish from vesiculated cell debris. However, chains of sporogonial stages were readily distinguished without the need for purification. Disrupted cells were therefore exposed to medium or medium containing one of the transporter inhibitors for 45 min and to calcein AM for an additional 15 min as above. The medium was then removed by centrifugation in a microcentrifuge and replaced with the HEPES-buffered solution as above. The cell suspension was then placed on the heated microscope stage, and the sporogonial stages were allowed to settle. Due to concerns that compounds such as polylysine might affect the membrane integrity of these small parasite stages (<2 m in width) the sporogonial chains were allowed to float freely. While there was some Brownian movement of these small parasite stages, because the chains averaged four cells at least one parasite cell was in focus in both the fluorescent and transmitted-light images at each observation. Infection assay. A mixture of uninfected and tests to determine the significance of differences between individual mean values. In experiments in which the levels of calcein fluorescence m-Tyramine hydrobromide of sporogonial stages were compared when the parasites were treated with carriers and with verapamil or cyclosporin A, Wilcoxon two-group rank tests were used to determine the significance of differences between means of replicate experiments. RESULTS Green monkey kidney cells were incubated with calcein AM, and their relative fluorescence was measured by confocal microscopy after removal of the probe from the medium. This fluorescence provided a measure of the intracellular concentration of the fluorescent calcein free acid which resulted from the removal of the acetoxymethyl groups from the calcein AM by cellular esterases. Calcein AM is extruded from cells by Pgp (1, 11), while MRP extrudes the free-acid form of this probe.