Roswell park memorial institute (rpmi)

Cardiomyocyte differentiation was induced using monolayer myocardial differentiation protocols as previously described (28 (link)), with minor modifications. Cells were treated with 12 μM CHIR99021 (SELLECK) for 1 day in RPMI (GIBCO) and B27 supplement minus insulin (GIBCO) (RPMI + B27-Insulin) until cells were expanded to 90% cell confluence and then replaced by RPMI+B27-Insulin. After 2 days, cells were treated with 5 μM IWP2 (TOCRIS). IWP2 was removed on day 6. From day 8, cells were cultured in RPMI + B27. Spontaneously contracting cells could be observed from day 10 to 14. Cells were replated for purification on day 15.

A coculture system of NCI-H441 cells and HPMECs was developed essentially as described previously (16 (link)). Briefly, Costar HTS Transwell 24-well plates (0.4-μm pore size; Sigma, Kawasaki, Japan) were coated with 0.1 mg/ml of isolated rat tail collagen I (Sigma) in 0.1 M acetic acid (Sigma) overnight at room temperature. Both sides of the membrane were coated. HPMECs were seeded in RPMI (Gibco) with 10% FBS and 1% penicillin-streptomycin (Gibco) at a density of 10 × 10⁵ cells/ml and incubated for 2 h to allow adherence to the membrane. Then, NCI-H441 cells were seeded at a density of 2.5 × 10⁵ cells/ml on the upper side of the membrane in RPMI (Gibco) with 5% FBS and 1% penicillin-streptomycin. Once the cells reached confluence (∼3 to 4 days), the medium was refreshed every 12 h. The upper compartment was refreshed with RPMI containing 5% FBS, 1% penicillin-streptomycin, 1 μM dexamethasone (Sigma), and 7 mM glucose (Gibco), while the lower compartment was refreshed with RPMI (5% FBS, 1% penicillin-streptomycin) and different concentrations of glucose (either 20 mM glucose every 12 h or alternating between 7 mM and 33 mM glucose every 12 h). Importantly, primary endothelial cells still retained their endothelial phenotype after culture in RPMI (data not shown). Once the TER across the membrane reached >1,000 Ω, the cells were infected with IAV.

Primary cultures of Vhl−/− ccRCC cells were obtained from surplus tumor specimens from surgeries of VHL patients. Written informed consent was obtained from all patients for the study of their samples. All procedures had been previously approved by the Ethics Committee in accordance with general accepted guidelines for human samples (075/2017).
Briefly, the collected fresh tissue from the excess of a resected RCC was placed in sterilized tubes containing ice-cold RPMI (GIBCO, Grand Island, NY, USA). Several PBS washing steps were done before specimens were chopped into ~1 mm³ pieces. The resulting tissue pieces were subjected to enzymatic digestion with [1 mg/mL] collagenase I and dispase II (GIBCO) in RPMI at 37 °C. After 45 min incubation, a gentle pipetting was performed for a complete disaggregation as well as a trypsin (GIBCO) digestion at 37 °C for 30 min. Then, minced samples were centrifuged and cell pellets were suspended in RPMI with 20% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (all from GIBCO) and incubated at 37 °C in humidity conditions. Sub-cultivation procedures followed a regular complete medium replacement (every 72 h or until the cultures were confluent).

To investigate which components are needed to induce motility of spz, the mosquitoes were dissected and the obtained salivary glands were crushed in eleven different formulations: (1) Phosphate buffered saline which contains potassium, sodium, chloride and phosphate (PBS; Life Technologies Inc.), (2) Hanks’ balanced salt solution which contains besides the salts present in PBS also glucose, calcium, magnesium, sulphate and bicarbonate (HBSS; Life Technologies Inc.), (3) Roswell Park Memorial Institute medium (RPMI; Life Technologies Inc.) which contains besides the components of HBSS also amino acids and vitamins, (4) PBS enriched with 3.5 mg/ml bovine serum albumin (BSA; Sigma-Aldrich), (5) HBSS + 3.5 mg/ml BSA, (6) RPMI + 3.5 mg/ml BSA, (7) PBS enriched with 10% fetal bovine serum (FBS; Life Technologies Inc.), (8) HBSS + 10% FBS, (9) RPMI + 10% FBS, (10) RPMI without amino acids (Caisson Labs) + 3.5 mg/ml BSA and (11) RPMI without glucose (Life Technologies Inc.) + 3 mg/ml BSA. All eleven formulations had a physiological pH of 7.0–7.5 and a viscosity of < 1.1 cP. For imaging of the spz, 10 µl of the spz solution was pipetted on the cover slip of a confocal dish without any precoating (ø14 mm; MatTek Corporation), covered with another cover slip (ø12 mm; VWR Avantor) and imaged within 45 min. The set-up is schematically depicted in Additional file 1: Fig. S1.