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. 2018 Nov 14;7:e41115. doi: 10.7554/eLife.41115

Figure 5. Comparative uptake of fluoroquinolones into human blood derived lymphocytes, neutrophils, macrophages and foamy macrophages, and into A549 epithelial cells.

(A) Intracellular to extracellular concentration ratios of MXF, LVX and GTX in the major cell types present in the cellular rim of necrotic lesions. Data were analyzed using the Friedman test (all means are significantly different from each other): *p<0.05, **p<0.01; (B) FACS analysis of Nile Red stained human bone marrow derived macrophages showing higher frequency of stained cells in macrophage populations stimulated with heat-inactivated M. tuberculosis (iMtb) compared to unstimulated macrophages. The percentage of Nile Red high cells is indicated. (C) Intracellular/extracellular drug concentration ratio of MXF, LVX, and GTX in unstimulated bone marrow derived macrophages (black bars, (M), and in iMTB stimulated foamy macrophages (green bars, FM), derived from seven individual donors (raw data in Figure 5—source data 1). Data were analyzed using the Wilcoxon matched-pairs signed rank test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5—source data 1. Raw data of fluoroquinolone accumulation in macrophages and foamy macrophages obtained from the blood of 7 individual human donors.
elife-41115-fig5-data1.xlsx (9.5KB, xlsx)
DOI: 10.7554/eLife.41115.020

Figure 5.

Figure 5—figure supplement 1. Ratio of fluoroquinolone uptake in foamy macrophages relative to non-foamy macrophages isolated from seven individual blood donors.

Figure 5—figure supplement 1.

Figure 5—figure supplement 2. Workflow for isolation and purification of lymphocytes and monocytes from donated packed leukocytes.

Figure 5—figure supplement 2.

Packed Leukocytes are carefully pipetted over a layer of Ficoll in 50 ml falcon tubes, and centrifuged at 1600 rpm/530 G for 30 min with no brake. PBMC layer is extracted with a pipette and washed 2 times in PBS and the final pellet is re-suspended in RPMI to a concentration of 108 cells/mL. EasySep CD14 antibody solution is added at 100 μL/10^8 cells. Incubation at room temperature for 15 min followed by EasySep magnetic beads at 50 μL/108 cells. After 10 min incubation to allow antibody binding to beads, the tube is placed in the magnet for 5 min. CD14+ cells (monocytes) migrate to the walls of the tube leaving only CD14- cells (lymphocytes) in suspension. While the tube remains in the magnet, the supernatant is poured off and collected. The tube is removed from the magnet, RPMI added, pipetting up and down to resuspend CD14+ cells, and the tube is returned to the magnet. The process is repeated 3 times.