Figure 4 | Scientific Reports

Figure 4

From: The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay

Figure 4

Calcein-AM efflux assay in the presence of an MTNP-generated indomethacin gradient.

(a) Fluorescence image of trapped Jurkat T cells taken 90 min after start of experiment. For analysis, the trap chamber was divided into 10 equal-area radial zones based on laminar flow modeling, with each zone having 2.22 μM greater indomethacin concentration than the one below. Zone 10 contained a maximum indomethacin concentration of 20 μM. Zone 1 had no drug, only plain media. Scale bar, 110 μm. (b) Fluorescence intensity vs. time of cells exposed to the indomethacin gradient for 70 min after loading with calcein-AM dye for 20 min. Images were taken every 5 min. Fluorescence was normalized to background illumination. Z1–Z10 represent the average fluorescence in arbitrary units of all stably trapped cells in each zone with an average of 30 cells per zone. The 95% confidence interval was derived after zeroing all cell traces at t = 0 and is given for the three bolded zones (Z9, Z5 and Z3), although the values were similar for all zones. The maximum 95% confidence intervals of all 10 zones are equal to or less than those given for Z3. Z9* (green line) represents a single outlier cell trapped in zone 9 whose fluorescence was more than 4 standard deviations lower than the other cells in zone 9 for the majority of its data points. (c) Plot of fluorescent intensity vs. time derived from the model based on the cellular enzymatic and transport kinetics depicted in the inset. Here, CAM is intracellular calcein-AM concentration, k1 is the rate of calcein-AM uptake by cells, k2 is the rate of calcein-AM conversion into calcein-F (CF) and k3f and k4f are the rates of calcein-AM and calcein-F efflux from cells, respectively. Both k3f and k4f are decreased in the presence of indomethacin. Best-fit parameters are reported in the text.

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