The painful and sensitive ATPase activity of ABCG2 in cell membrane prepared from High Five insect cells was measured by using the BD Gentest ATPase assay kit according to the manufacturers guidelines. Propidium iodide at a final concentration of 2 g/mL was included with the cells to entrance viable cells. The cells were filtered via a 40 m cell strainer to obtain a single cell suspension before searching. Explanations and sorting were done with fluorescence activated cell sorting. PFT The Hoechst 33342 dye was thrilled at 357 nm and its fluorescence was dual wavelength reviewed. Tumorigenicity Experiments Sorted SP and non SP cells from A549 cells were subcutaneously injected into the NOD/SCID mice. Groups of mice were inoculated with SP or low SP cells at 103. The mice were killed 44 d after tumor cell injection. Detection of Cell Surface Expression of ABCG2 and ABCB1 by Flow Cytometer SP cells were collected and washed 3 times using an isotonic PBS buffer. For ABCG2 phrase investigation, APC conjugated anti human Bcrp1/ABCG2 reagent were mixed with 25 L of Hamilton academical blocked cells. After incubating for 45 min at 4 C, the cells were washed twice with PBS buffer and re-suspended in 400 D PBS buffer for flow cytometric analysis. Isotype control samples were treated Latin extispicium in a similar fashion with allophycocyanin labeled mouse immunoglobin G2b antibody. For ABCB1 flow cytometric evaluation, 106 cells were incubated at 4 C for 30 min with 10 L of CD243 PE conjugated antibody, cells were then washed and re-suspended in PBS. Isotype control samples were treated with mouse IgG2a antibody in parallel. Controls and tests were assessed with a flow cytometer. Apoptosis Assay Cells were seeded onto a six well plate at a density of approximately 105 cells/well. After treatment with different concentrations of axitinib in the presence of 0. 2 mol/L topotecan map kinase inhibitor or mitoxantrone for 48 h, both connected and floating cells were collected and washed with ice cold PBS twice. Cells were resuspended in 100 L of binding buffer, and the Alexa Fluoro 488 annexin V and propidium iodide were added before incubation at room temperature for 15 min. Following the incubation period, we added 400 L 1 binding load, mixed gently and analyzed via FACS. Doxorubicin and Rhodamine 123 Accumulation The effect of axitinib on the intracellular accumulation of rhodamine and Dox 123 was done as previously described. Fleetingly, the cells were treated with axitinib of numerous concentration or car at 37 C for 3 h. Consequently, 10 mol/L Dox or 5 mol/L rhodamine 123 was added and the incubation was continued for yet another 3 h or 0. 5 h, respectively. The cells were then collected, centrifuged and washed three times with cold PBS. Finally, the cells were analyzed with flow cytometric analysis. FTC was used as a control inhibitor of ABCG2 in S1 and S1 M1 80 cells.