Express five medium

ED cells were grown in Iscove’s modified Dulbecco’s medium (IMDM; Pan, Biotech, Aidenbach, Germany) containing 20% fetal bovine serum (FBS; Biochom GmBH, Berlin, Germany), 1 mM sodium pyruvate (Pan Biotech, Aidenbach, Germany), 1% nonessential amino acids (NEAA; Biochom GmBH, Berlin, Germany), and P-S solution (100 U/ml penicillin: Panreac, AppliChem GmBH, Darmstadt, Germany); 100 μg/ml streptomycin: Alfa Aesar, Thermo Fisher Scientific, Kandel, Germany (P-S) at 37°C under a 5% CO₂ atmosphere.
Human embryonic kidney (293 T, ATCC CRL-11268) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Biochom GmBH, Berlin, Germany), supplemented with 10% FBS and P-S. Sf9 cells (IPLB-Sf21-AE, Invitrogen, Germany) were propagated in serum free Sf-900 III medium (Gibco, Thermo Fisher Scientific, New York, USA) and High Five™ cells (BTI-TN-5B1-4, Invitrogen, Germany) in serum free Express Five medium (Gibco, Thermo Fisher Scientific, New York, USA) at 27°C on orbital shaker.

All cells were in the D-Mel2 background and were cultured in Express Five medium (Invitrogen) supplemented with glutamine, penicillin, and streptomycin. All stable cell lines were selected in medium containing 20 µg mL⁻¹ blasticidin. Expression of the copper-inducible transgenes were induced with CuS0₄ (300 μM) for at least 8 h. For Aurora B inhibition, cells were treated with 20 μM Binucleine 2 (EMD Millipore) for 1 h before being processed for time-lapse microscopy. For Polo kinase inhibition, cells were treated with BI 2536 (MedChem Express) for 1 h before being processed for time-lapse microscopy or IF. We used different concentrations of BI 2536 depending of the experiment (30 nM, 100 nM, 200 nM, 1 μM). For Cdk1 inhibition, cells were treated with 10 μM RO 3306 (Tocris Bioscience) for 1 h before being lysed.

At temperature of 27°C, Trichoplusia ni cell line High Five (Invitrogen, USA) was cultured in Express Five medium (Invitrogen, USA) with 1.8 mM/mL l-glutamine (Invitrogen, USA) were cultured. The cells were co-transfected with a synthesized miRNA mimic (300 nM) and pIZ/EGFP consisting of 3′UTR of wsv459 or wsv322 (6 µg/mL) at about 70% confluence. The miRNA mimic was synthesized by T7 Kit siRNA Synthesis (TaKaRa, Japan). All experiments of transfection were accomplished with Cellfectin (Invitrogen) according to Cellfectin’s manual in triplicate. 12 h for after culture, cells were planked with cell density of 2.0 × 10⁴ cells/well to 96-well plates. The fluorescence density of High Five was detected using a Flex Station II microplate reader (Molecular Devices, USA) with condition of Ex (490 nm)/Em (510 nm) at 48 h post-transfection. The fluorescence value was revised by subtracting the spontaneous fluorescence of non-EGFP cells.

Artificial antigen presenting Drosophila cells (aAPC) were cultured in Express Five Medium (Invitrogen) supplemented with 2mM L-glutamine (Invitrogen) and 200ug/ml Geneticin (Invitrogen) with shaking (100rpm). The cells were cultured every 2–3 days until the viability was >85%. Cells were then washed with PBS, re-suspended in media +5μg/ml UVADEX (Johnson & Johnson) and subjected to cross-linking for 10 minutes at 7.7 Joules/ cm² in a VueLife bag (American Fluoroseal Corporation). An ILT72 UVA Radiometer (Life Technologies) was used for the cross-linking. aAPC cells were loaded with a melanoma antigen recognized by T cells (MART-1) peptide (CS Bio, Menlo Park, CA) by incubating 20 x 10⁶ cells (1 x 10⁷ cells/ml) in Express Five media + 5ug/ml Beta-2M (Janssen in-house) and 0.1μg/ml MART-1 peptide for 4 h at room temperature with mixing every 30 minutes. CD8⁺ T cells were incubated at 37°C for 6 days with Mart-1 peptide loaded aAPCs (1:10 ratio of aAPCs: T cells) and 25ng/ml IL-21 (PeproTech, Rocky Hill, NJ). On day 6, 20 U/ml of IL-2 and 30U/ml IL-7 (PeproTech, Rocky Hill, NJ) were added to the cells and incubated for an additional 2 days. On day 8, re-stimulation was restarted following the procedure stated above and continued until day 14. Flow cytometric analysis was performed to determine viability and TIM-3 receptor expression.

All cells were in the D-Mel (d.mel-2) background and were cultured in Express Five medium (Invitrogen) supplemented with glutamine, penicillin and streptomycin (Wisent). Transfections were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) following the manufacturer's instructions. All stable cell lines were selected in medium containing 20 µg ml⁻¹ blasticidin. While inducible pMT-based vectors contain the blasticidin resistance gene, pAc5-based vectors were co-transfected with pCoBlast to confer blasticidin resistance to the cells. Expression of the copper-inducible transgenes was induced with CuSO₄ (300 µM unless otherwise indicated) for at least 8 h before experiments.
For RNA interference, dsRNAs were generated from PCR amplicons using a T7 RiboMAX kit (Promega). dsRNA derived from the bacterial kanamycin resistance gene was used as a non-target control. Twenty milligrams of dsRNA was transfected in 1X10⁶ cells in a well of a 6-well plate using Transfast transfection reagent according to the manufacturer's protocol.

All cells were in the D-Mel (d.mel-2) background and were cultured in Express Five medium (Invitrogen) supplemented with glutamine, penicillin and streptomycin (Wisent). Transfections were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) following the manufacturer's instructions. All stable cell lines were selected in medium containing 20 µg ml⁻¹ blasticidin. While inducible pMT-based vectors contain the blasticidin resistance gene, pAc5-based vectors were co-transfected with pCoBlast to confer blasticidin resistance to the cells. Expression of the copper-inducible transgenes was induced with CuSO₄ (300 µM unless otherwise indicated) for at least 8 h before experiments.
For RNA interference, dsRNAs were generated from PCR amplicons using a T7 RiboMAX kit (Promega). dsRNA derived from the bacterial kanamycin resistance gene was used as a non-target control. Twenty milligrams of dsRNA was transfected in 1X10⁶ cells in a well of a 6-well plate using Transfast transfection reagent according to the manufacturer's protocol.