Various experimental procedures for the calcein-AM efflux assay are described in literature (Homolya et al. 1993, Hollo et al. 1994, Feller et al. 1995, Liminga et al. 1994, Legrand et al. 1998). They differ in several parameters such as incubation time, incubation temper-ature, calcein-AM concentration and cell number in the sample. The calcein-AM loading concentration of the cells is depending on the used calcein-AM concentration and the number of cells per sample. Homolya et al. (1996) examined the appropriate conditions for the calcein-AM efflux assay in great detail and found that at calcein-AM concentra-tions between 0.1 to 1µM and cell numbers between5·104 ml−1 and 1·106 ml−1, clearly distinguishable fluorescence differences between p-gp expressing cells and cells without a MDR1 phenotype can be detected.
In our department a fluorescence assay was established for the determination of
intra-4.4 Optimization of the calcein-AM efflux assay for flow cytometry 79 cellular calcium using fluo-4 at the flow cytometer (Schneider et al. 2002). Calcein-AM is analogous to fluo-4. Fluo-4, an acetoxymethylester, can easily penetrate the cell mem-brane. Inside the cell the ester bonds of fluo-4 are cleaved by intracellular esterases and the resulting fluo-4 can complex intracellular calcium. Due to the similar uptake and transformation mechanism of both fluo-4 and calcein-AM the loading procedure of the fluo-4 assay can be adapted to the calcein assay using Pluronics F-127 to improve the solubility of calcein-AM and its penetration into the cells (Granados et al. 1997).
To optimize the aforementioned assay conditions for the calcein-AM efflux assay several experiments were carried out using Kb-V1 cells (wildtype) which are negative for the MDR1 phenotype. In a first series of experiments the incubation time and the incubation temperature were altered (Fig. 4.4 and Fig. 4.5). Subsequently, the optimal calcein-AM loading concentration was determined by incubation of cells with different calcein-AM concentrations ranging from 0.5 to 2.0 µM under the optimized conditions described above (Fig. 4.6).
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Figure 4.4: Optimization of calcein-AM efflux assay conditions by variation of incubation times using Kb-V1 wildtype cells (10 min - solid line, 30 min - dashed line, 60 min - dotted line). All experiments were carried out at 25 ℃ using a cell number of1·106 ml−1 and a calcein-AM concentration of 1.25 µM.
In Fig. 4.4 the results of the calcein-AM assay carried out at 10, 30 and 60 min, respectively, are displayed in histograms. The cell associated fluorescence increased with longer incubation times. However, the fluorescence intensity measured after an incubation time of 10 min is even high enough for p-gp activity measurements compared to that after
80 Establishment and application of a calcein-AM efflux assay
Figure 4.5: Optimization of calcein-AM efflux assay conditions by variation of incubation tem-peratures using Kb-V1 wildtype cells (37 ℃ - solid line, 25 ℃ - dashed line). All experiments were carried out using a cell number of 1·106 ml−1, a calcein-AM concentration of 1.25µM and an incubation time of 10 min.
a 60 min incubation. Therefore, the calcein-AM efflux assay can be performed with an incubation time of 10 min. In a next step, the incubation temperature was varied (Fig.
4.5). The cell associated fluorescence after an incubation at 37 ℃ was similar compared to the fluorescence intensity after an incubation at 25 ℃. As with an incubation at 37 ℃
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Figure 4.6: Optimization of calcein-AM efflux assay conditions by variation of calcein-AM load-ing concentrations usload-ing Kb-V1 wildtype cells. All experiments were carried out at the optimized incubation conditions using a cell number of1·106 ml−1.
4.4 Optimization of the calcein-AM efflux assay for flow cytometry 81 physiological conditions can be simulated, this temperature was chosen for the calcein-AM incubation.
After the optimization of the incubation conditions the appropriate calcein-AM concen-tration was determined (Fig. 4.6). The fluorescence intensities increased with increasing calcein-AM concentrations. The maximum fluorescence intensities did not differ very much. However, the distribution of the fluorescence intensities after an incubation with 0.5 and 0.75 µM, respectively were relatively broad compared to incubation with higher calceAM concentrations. Thus, in order to get a narrow distribution of fluorescence in-tensities and to use only a minimum of calcein-AM, the optimal calcein-AM concentration was determined to 1.0 µM.
Analyzing the calcein-AM efflux in Kb-V1/VBL cells, that are a p-gp overexpressing subclone of the Kb-V1 cell line, a much lower cell assiciated fluorescence intensity was detected compared to the fluorescence intensity of calcein loaded Kb-V1 cells due to the p-gp mediated efflux of calcein-AM before its transformation into calcein. Both signals were well separated from each other (Fig. 4.7). Hence, Kb-V1/VBL cells provide a system that can be used for the examination of substances with respect to their influence on p-gp mediated efflux. Kb-V1 wildtype cells serve as a positive control for the p-gp mediated calcein-AM efflux. Using the determined parameters for the calcein-AM assay MDR1
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Figure 4.7: Calcein-AM efflux assay with Kb-V1 (dotted line) and Kb-V1/VBL (dashed line) cells carried out at the optimized assay parameters (mixed population - solid line;
10 min, 37 ℃, 1µM calcein-AM, 1·106 ml−1 cells)
82 Establishment and application of a calcein-AM efflux assay positive and MDR1 negative Kb-V1 cells could be even detected with this assay if they are examined in a mixed cell population (Fig. 4.7).
In summary, the appropriate conditions for the calcein-AM efflux assay to identify p-gp expression in cells and to investigate p-gp substrates and inhibitors are an incubation temperature of 37 ℃, an incubation time of 10 min and a calcein-AM concentration of 1 µM using 1·106 cells ml−1 in each sample.