Fcs5 express software

Flow cytometric analysis was carried out on leukemic cells and on cell suspensions (10⁶ cells/mL) of colon carcinoma cells obtained by incubating monolayer cell cultures with EDTA and trypsin. For the P-gp determination on the cell surface, the cells were incubated for 30 min at RT with MAb FITC conjugate CD243. After washing with ice-cold phosphate buffer saline (PBS, pH 7.2) with 1% bovine serum albumin (BSA, Sigma), cells were immediately analyzed. Dead cells were excluded from the analysis by gate on cells size before the acquisitions. Ten thousand events were acquired and fluorescence was analyzed with a FACScanto flow cytometer (Becton Dickinson) equipped with a 15 mW, 488 nm, air-cooled argon ion laser. The fluorescence emissions were collected through a 530 nm band-pass filter for fluorescein.
For quantitative evaluation of P-gp expression, the mean fluorescence channel (MFC) was calculated by FCS5 express software (De Novo software). P-gp content in arbitrary units was expressed as the ratio of the MFC of labeled resistant cells on the MFC of parental ones.

To evaluate the immune phenotype, PC3 and PC3RR cells were detached with 0.25% trypsin EDTA (Sigma), washed with PBS and incubated with bv421-conjugated Integrin alpha-2 antibody (BD Bioscience, San Jose, CA, USA) in PBS/0.1% BSA (Sigma) for 30 min on ice prior to flow cytometric analysis. Propidium iodide (PI) solution (Sigma-Aldrich P4864) was added to exclude dead cells. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FCS5 Express Software (De Novo Software, Glendale, CA, USA). For cell cycle analysis, PCa and PCaRR cells were fixed with 70% ethanol, washed three times with PBS and stained for 3 h at room temperature with PI, then analyzed using flow cytometry.
To assess the effects of Docetaxel treatment, PC3, PC3RR, DU-145 and DU-145RR cells were seeded 9 × 10⁴/well and 6 × 10⁴/well, respectively, in 12-well plates, then treated with different concentrations (from 10 nM to 100 nM) of Docetaxel (Sigma-Aldrich) for 24 and 48 h. Then, apoptosis/necrosis were assayed by using PI/Annexin Pacific Blue staining (Thermo Fisher Scientific, Rockford, IL, USA) and evaluated using flow cytometry.

Cells were detached and incubated with APC-conjugated CD44 antibody (BD-PharMingen, San Diego, CA, USA 559942) or isotype control APC-conjugated IgG2B (BD-PharMingen) in PBS BSA 1% (Sigma) for 30 minutes on ice prior to flow cytometric analysis. Sytox Blue Stain (Life Technologies, Eugene, OR, USA S34857) was added to exclude dead cells. For CD44v8-10 staining 1×10⁵ cells were incubated with 3 μg/ml anti-human CD44v9 primary antibody (clone: RV3) (Cosmo Bio Co. Ltd, Tokyo, Japan LKG-M001) in PBS 0.2 % BSA for 45 minutes at 4°C. Cells were then incubated with the secondary antibody APC-labeled Goat anti-Rat IgG (H+L) (Invitrogen, Carlsbad, CA, USA A10540) for 30 minutes at 4°C. After washing, cells were assayed using a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data analyzed employing FCS5 express Software (De Novo Software). For cell cycle analysis, after labeling for CD44v9, the cells were fixed with 70% ethanol, a few drops at a time mixing the cells, and incubated on ice for 30 minutes. The cells were centrifuged at 500 x g for 10 minutes, washed once in PBS by centrifugation and resuspended in 1 ml PBS containing 5 μg/ml of propidium iodide. Samples were assayed by CyAn ADP flow cytometer and analyzed employing FCS5 express Software.