Rpmi 1640 medium

Mice were sacrificed by CO₂ asphyxiation followed by cervical dislocation, and the spleen and lungs were aseptically removed. The spleens were homogenized using the plunger of a 3-mL syringe (Becton Dickinson) through a 100-μm strainer and washed with RPMI-1640 medium (GenDEPOT). The lungs were minced using sterile scissors and digested in 5 mL RPMI containing 3 mg/mL Collagenase A (Sigma) and 0.15 mg/mL DNase I (Sigma) at 37 °C for 1 h. Digested lungs were homogenized using the plunger of a 3-mL syringe (Becton Dickinson) and filtered through a 40-μm strainer (EZFlow Cell Strainer, Foxx Life Sciences). Red blood cells were lysed using 0.83% ammonium chloride. Cells were washed and resuspended with complete RPMI (RPMI 1640, 5% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin). The total number of live cells isolated was determined by trypan blue exclusion staining. The absolute number of cell populations were calculated based on the numbers of splenocytes/leukocytes from the lungs and the percentages of populations as determined by FACS.

Cells were incubated at 37 °C in a humidified chamber at 5% CO₂. Lung cancer cells (A549, H441, and H358) were cultured in RPMI-1640 medium (GenDEPOT, TX, USA) and other cells (HPNE-P2M, HPNE-Kras, PNAC1, MCF7, MDA-MB-231, MDA-MB-468, DB7, and BT549) were cultured in DMEM medium (GenDEPOT). All media were supplemented with 10% FBS medium (GenDEPOT) and 1% penicillin–streptomycin medium (GenDEPOT).
Lipofectamine RNAiMAX (Invitrogen, CA, USA) was used for RNA-oligonucleotide transfection, whereas plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. All oligonucleotides were purchased from Sigma (MO, USA). The sequence of each gene is shown in follows. RORA and RORC siRNA are siRORA: 5′-CAAGAUCUGUGGAGACAAAdTdT and siRORC: CGAGGATGAGATTGCCCTCTAdTdT, respectively. For negative control, MISSION® siRNA Universal Negative Controls #2 (Sigma, MO, USA) was used.
RORA and RORC CRISPR knockdown cells were generated as previous described [40 (link)]. Briefly, sgRNAs were designed by using the CPRISRdirect software (https://crispr.dbcls.jp/). MDA-MB-231 cells were transfected with RORA and RORC sgRNA constructs and selected by puromycin. Colonies were expanded and validated for knockdown efficiency. Experiments were performed by using cells at early passages (before p5).