Rpmi medium

The human CRC cell line HCT-116 was obtained from American Type Cell Collection (ATCC, Manassas, VA, USA), and maintained in RPMI medium (ATCC) supplemented with 5% heat inactivated fetal bovine serum (FBS; Mediatech, Herndon, VA, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in a humidified incubator containing 5% CO₂ and 95% air at 37 °C, kept sub-confluent, and medium was changed every other day. All cells were assayed within 5–25 passages. Enrichment culture of tumor-derived colon-spheres was performed by incubating parental HCT-116 cells in serum-free medium (SFM) composed of DMEM/F-12 medium supplemented with 2% B-27 supplement, 20 ng/mL recombinant human epidermal growth factor, 10 ng/mL fibroblast growth factor-basic (Life Technologies, Grand Island, NY, USA), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 10 μg/mL insulin (Sigma-Aldrich) in ultra low-attachment plates (Corning, Lowell, MA, USA) at 37 °C. Plated under these anchorage-independent conditions in supplemented-SFM, tumor cells form floating spheres reported to represent the growth of CSC [27 (link),31 (link),54 (link)].

The human RC13 ARMS (SJCRH30 [RC13, RMS 13, SRJH30] (ATTC CRL2061) [17 (link), 37 (link), 38 (link)] and RD ERMS (ATCC CCL-136) [39 (link)–41 (link)] cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). RC13 and RD cells were plated on tissue culture dishes and grown in RPMI medium (ATCC) and Dulbecco’s minimal essential medium (DMEM), respectively, containing 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY). Cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% Triton X-100 prior to labeling with antibodies as described below.

The LNCaP and CWR22Rv1 cells were cultured in RPMI medium (ATCC, Manassas, VA). PC-3 and VCAP cells were grown in the F-12K medium (ATCC, Manassas, VA). All medium was supplemented with 10% fetal bovine serum (FBS, ATCC) and 1% antibiotic solution (Sigma-Aldrich, St. Louis, MO). Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. Cells were grown as monolayers and were harvested or split when they reached 80% confluence to maintain exponential growth. One day before the cell binding experiment, PCa cells were seeded in 24-well plates.
All animal experiments were conducted in accordance with standards of the Institutional Animal Care and Utilization Committee of Nanjing Medical University. 5 × 10⁶ PC-3, LNCaP, CWR22Rv1, or PCa cells in 50% Matrigel (Becton Dickinson, Heidelberg, Germany) were injected subcutaneously into the mice left armpit to establish animal models.

Primary human monocytes were isolated from 125 mL of human blood obtained from the Gulf Coast Regional Blood Center (Houston, TX). Blood was diluted in RMPI medium and separated by density gradient separation on Ficoll at 2000 rpm for 20 min. The plasma was removed from the separated sample and the buffy coat was collected. Buffy coat was diluted with DPBS containing 2% FBS and 1 mM EDTA and centrifuged at 1500 rpm for 15 min. The supernatant was removed, and all cells were combined and mixed carefully. Combined cells were then centrifuged at 1500 rpm for 10 min, and the supernatant was removed. Cells were resuspended into 1 mL of Dulbecco’s PBS (DPBS) containing 2% FBS and 1 mM EDTA. Cells were then diluted to 5 × 10⁷ cells/mL, and monocytes were separated by the EasySep Human Monocyte Enrichment kit without CD16 depletion (Stemcell number 19058) according to the manufacturer's protocol. Primary human monocytes were then cultured in RPMI medium (ATCC) containing 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/liter glucose,1500 mg/liter sodium bicarbonate, supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C in 5% CO₂ atmosphere.

Human distal lung epithelial cell line NCI-H441 was obtained from the American Type Culture Collection (ATCC) and cultured as previously described [16 (link)]. H441 cells were grown in RPMI medium (ATCC) containing 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). For Ussing chamber assays, cells were seeded on permeable support filters (Costar) at a supraconfluent density (∼5 × 10⁶ cells/cm²) and incubated in a humidified atmosphere of 5% CO₂ at 37°C. Dexamethasone (250 nM, Sigma) was supplemented to stimulate ENaC expression. Cells reached confluency in the Costar Transwells 24 h after plating. At this point, media and nonadherent cells in the apical compartment were removed to adapt the cells to the air-liquid interface culture. Culture media in the basolateral compartment were replaced every other day, whereas the apical surface was rinsed with PBS. Transepithelial resistance was measured by an epithelial tissue volt-ohm-meter (World Precision Instruments). Highly polarized tight monolayers with resistance >500 Ω cm² were used for measuring short-circuit current (I_sc) levels of transepithelium.

Jurkat cells (Jurkat, Clone E60-1, ATCC: TIB152™) were cultivated in RPMI medium (cat. no. 72400-021; Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Electroporation was performed using a NEPA21 electroporator (Nepa Gene) with a total amount of 20 μg DNA. Cells were induced 24 h after electroporation with 1 μM rupintrivir and kept for a further 24 h before determining the mean fluorescence intensity (MFI) of citrine with a CytoFLEX S flow cytometer (Beckman Coulter).