Facs aria high speed cell sorter

Cell lines stably expressing WT, K433M, K1076M, and K433M/K1076M double mutant MDR1/P-glycoprotein were established by the Sleeping Beauty (SB) transposon-based gene delivery system, using the 100 fold hyperactive SB transposase68 (link). NIH 3T3 mouse fibroblast and MDCK II canine kidney cells were co-transfected with the SB transposase and SB transposon vector constructs by Lipofectamine2000 reagent (Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. Briefly, 3 × 10⁵ cells were seeded in 6-well-plates, 24 hours later cells were transfected with 2 μg vector DNA per well in a 10:1 ratio for the SB transposon and transposase constructs. 48 hours after transfection transgene positive cells were sorted by flow cytometry (FACS Aria High Speed Cell Sorter, Becton Dickinson) based on the cell surface expression of wild-type and mutant MDR1/P-glycoprotein. Protein expression was measured by antibody labeling using the human MDR1/P-glycoprotein specific monoclonal antibodies MRK16 (Abnova GmbH, Heidelberg, Germany) or 15D3. To obtain homogenously expressing cell populations, sorting procedure was repeated 2 weeks after transfection.

Cardiomyocytes from newborn rats was prepared by collagenase II digestion and cultured in DMEM/M199 medium with 10% FBS, 100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.1 mM of Brdu (Sigma, St. Louis, MO, USA). C-kit(+) CSCs were purified by staining with PE-conjugated anti-c-kit monoclonal (#553355, BD) and sorting with a FACS Aria high-speed cell sorter (Becton Dickinson, San Jose, CA, USA), cultured in DMEM-Ham’s F-12 containing 10 ng/mL of EGF (PeproTech, Princeton, NJ, USA), 20 ng/mL of bFGF (PeproTech), 10 ng/mL of LIF (PeproTech), 10% of ES-cell qualified FBS (Gibco, Carlsbad, CA, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL). The cardio fibroblasts were removed from cardiomyocytes through differential adhesion.

HeLa cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, cat. #31966047) supplemented with 10% of fetal bovine serum (cat. #10500064), 1% of L-glutamine (cat. #25030081), and 1% of penicillin/streptomycin (cat. # 15070063, all from Thermofisher Scientific) using standard cell culture methodology. Cells were transfected with FuGENE^® HD reagent (Roche Applied Science, Penzberg, Germany) in a 6-well or 24-well plate, according to the manufacturer’s instruction. To stain the cell nuclei, 10 μM of the Hoechst 33342 dye was used according to the standard protocol. EGFP and Hoechst fluorescence was detected by an IX51 fluorescence microscope (Olympus, Shinjuku City, Tokyo, Japan).
To establish cell lines stably expressing EGFPm-mirtron constructs, we applied the Sleeping Beauty transposon-based gene delivery technology as described earlier [51 (link)]. Following transfection, cells were sorted for EGFP positivity at day 8 and subsequently at day 15 using a FACS Aria High Speed Cell Sorter (Beckton-Dickinson, Franklin Lake, NJ, USA) to obtain homogenously expressing cell populations. Stable expression was also checked by subsequent FACS analyses. The established cell lines were further used for mRNA level and western blot experiments.

Control PCa cells (PC3^Control or DU145^Control) or GAS6 overexpressed PCa cells (PC3^GAS6OE or DU145^GAS6OE) (1 × 10⁵) were seeded onto 12-well culture plates, and then cultured for 48 hours. PCa cells were incubated with PE-anti-CD133 antibody (cat. 130-080-901, Miltenyi Biotec, San Diego, CA) and APC-anti-CD44 antibody (cat. 559942, BD Biosciences, San Jose, CA) for 20 min at 4°C. The CD133+/CD44+ fraction was analyzed with a FACS Aria High-Speed Cell Sorter (BD Biosciences).

Pre-Th1 and pre-Th2 CD25^– memory and CD25⁺CD127^hi memory cells were identified using the antibodies described in the section above. The 4 cell populations were sorted on a BD FACSAria high-speed cell sorter. Gates used to sort cell subsets are shown in Supplemental Figure 4.