Facs diva cell sorter

Three different GIC cultures were used to establish PBK knockdown cell lines each with three different shRNA constructs (RMM4431-98972202, RHS4430-101071930, RHS4430-101072940, Thermo Scientific, Open Biosystems). Nine μg of plasmid DNA were transfected into the 293FT cell line using Arrest-In transfection reagent (according to the manufacturer’s protocol). Briefly, viral supernatants collected after 48 and 72 h were centrifuged at 3000 rpm for 20 min at 4 °C and filtered through a sterile 0.45 μm low protein binding filter (Sarstedt). The virus was then concentrated in sterile SW28 ultracentrifuge tubes by ultracentrifugation (Beckman Optima™ LE-80 K ultracentrifuge) equipped with a SW-28 rotor at 23,000 rpm for 1.5 h at 4 °C. The pellet was resuspended in 200 μl of DMEM and aliquots of the concentrated virus were stored at −80 °C. Tumor cells (10⁵ cells/well) were then transduced in 24 well plates by adding 10 μl concentrated virus/well and the cells were incubated for 48 h at 37 °C in 5 % CO₂. Three to five days after transduction, GFP positive cells were purified by FACS sorting using a FACS Diva cell sorter equipped with an argon ion laser, ‘TurboSort Plus’ option, and Diva software (Becton Dickinson). The cells were then selected in 2 μg/ml Puromycin (Sigma, USA) for 3–4 weeks and used for functional assays.

We evaluated the self-renewal capacity of RCC-41-PDX-1/CD132⁺, RCC-41-PDX-2, RCC-41-PDX-2/CD133⁺, and RCC-41-PDX-2/CD133⁻ by performing a limiting dilution assay for spheroid formation. Cells were plated using a FACS DiVa cell sorter equipped with autoclone software (Beckton Dickinson, Le Pont-de-Claix, France) at a density of one and 100 cells per well in ultralow attachment 96-well plates (Corning Life Sciences, Acton, MA). Each well was supplemented with 200 μl of serum-free complete DMEM-LG. After 3 weeks, each well was examined under a light microscope and the total number of wells containing spheroid colonies was determined. Five replicates were used for each condition, and the experiment was repeated two times.

The percentage of apoptotic cells induced by nisin treatment was determined by flow cytometry. Briefly, cells were detached by incubation with enzyme-free dissociation buffer (Invitrogen), pelleted by centrifugation, and stained with Annexin V (BD Pharmingen) for analysis by flow cytometry (FACSDiVa Cell sorter, Becton Dickinson).

For sorting cells, cells were dissociated with Accutase (Stem Cell Technologies, Vancouver, Canada), washed twice with PBS and processed for CD44 (Miltenyi Biotec, Cologne, German) and CD133 (Miltenyi Biotec, Cologne, German) multicolor staining, along with appropriate negative controls and single‐color positive controls. The CD44⁺/CD133⁺ populations were sorted out by a BD FACS Diva cell sorter (BD, USA).

FACS analyses were performed on BD FACSDiva Cell Sorter (BD FACSDiva Software, RRID:SCR_001456) at the Duke Flow Cytometry Core. For isolation of SP and non-SP cells, single-cell suspensions were treated with 2.5 mg/mL Hoechst 33342 dye (MilliporeSigma, B2261) alone, or in combination with 50 mmol/L verapamil (MilliporeSigma, V4629) as a negative control, for 90 minutes at 37°C. SP cells were identified using dual-wavelength analysis (blue, 424–444 nm; red, 675 nm) after excitation with 360 nm UV laser. To enrich for tumor SP cells, digested tumor mixture was stained with rat anti-mouse CD45-PE-Cy7 (BD Biosciences, 552848, RRID:AB_394489) or rat anti-mouse CD45-APC-Cy7 (BD Biosciences, 557659, RRID:AB_396774) antibody at 1:800 dilution, or cells were sorted on the expressed fluorescent reporters. RFP and YFP cells from the KPCC tumors were identified using the blue 488 nm laser. Dead cells were eliminated with propidium iodide (Thermo Fisher Scientific), according to the manufacturer’s instructions. To analyze the purity of the sorted cells, we took a small sample of sorted cells immediately after FACS and reanalyzed for the presence of different sorted fractions using the same sorter (FACSDiva Cell Sorter, RRID:SCR_001456). Analysis of flow cytometry data were performed using FloJo (version 10.7).

Splenocyte (SC) suspensions were obtained by passing the mouse spleen through a 70-μm cell stainless steel strainer, and erythrocytes were depleted by hypotonic lysis. T cell-depleted SC (act as APC) were enriched from pooled spleens by positive selection via magnetic-activated cell sorting (MACS) using a Pan T Cell Isolation Kit (Miltenyi Biotec). CD4⁺ T cells were enriched from the same pool by negative selection via MACS using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). CD4⁺CD25⁻ T cells were sorted by antibody cell sorting on an FACS Vantage SE with a FACS Diva Cell Sorter (BD Bioscience) to >99 % purity.