Fitc conjugated anti p gp antibody

RPMI 1640 medium and DMEM (Dulbecco’s Modified Eagle Medium) were acquired from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). L-glutamine, trypsin, and penicillin–streptomycin solution were from Biowest (Nuaillé, France). Thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), DMSO, Hoechst 33342, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). An Apoptosis Detection Kit based on Annexin-V-FITC (AV) and propidium iodide (PI) staining was acquired from Abcam (Cambridge, UK). Bovine serum albumin (BSA) was obtained from Serva (Heidelberg, Germany). Rhodamine 123 (Rho123) and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma (St Louis, MO, USA). Carbonyl cyanide m-chlorophenyl hydrazine (CCCP, C2759) was purchased from Sigma-Aldrich (Darmstadt, Germany). FITC-conjugated anti-P-gp antibody was obtained from BD Biosciences (Winnersh, Berkshire, UK). Anti-P-gp mouse monoclonal antibody (Abcam, Cambridge, UK; ab10333) and secondary antibodies Alexa Fluor 555 goat anti-mouse secondary antibody (#A-21422, Thermo Fisher Scientific, Waltham, MA, USA) were obtained.

RPMI 1640 medium, fetal bovine serum (FBS), antibiotic-antimycotic solution, L-glutamine, and trypsin/EDTA were purchased from Biological Industries, Beit HaEmek, Israel. Rho 123, DMSO and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma–Aldrich Chemie GmbH, Hamburg, Germany. FITC-conjugated anti-P-gp antibody was purchased from BD Biosciences, Plymouth, UK. A Pgp-Glo^TM assay system was obtained from Promega, Madison, WI, USA.

To assess the P-glycoprotein expression level in MDR GBM cells, flow-cytometry was employed. U87-TxR cells seeded in adherent 6-well plates were treated with 5 µM Si306, pro-Si306 or dasatinib for 72 h. Then, cells were collected by trypsinization, washed in PBS, and then directly immuno-stained by FITC-conjugated anti-P-gp antibody (BD Biosciences, Winnersh, Berkshire, UK). To discriminate the level of background fluorescence, an isotype control IgG2bκ (Abcam, Cambridge, United Kingdom) was used. The fluorescence of FITC-conjugated anti-P-gp was analyzed on assessed on the fluorescence channel 1 (FL1) of the CyFlow Space Partec flow-cytometer (Sysmex Partec GmbH, Görlitz, Germany). After gating to exclude the cell debris and dead cells, a minimum of 10,000 cells per sample was assayed. Summit Dako Software was used for the analyses of results obtained by flow-cytometry.