Dulbecco s modified eagle s medium dmem

HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Paneco) supplemented with 2 mM L-Glutamine (Gibco), 10% fetal bovine serum (HyClone), 50 U/mL penicillin and 50 μg/mL streptomycin (both from Paneco). Transfection of HEK293 cells with H1N1 HA-encoding mRNA was performed as described previously (21 (link)) with minor modification. Briefly, HEK293 cells were plated on a 12-well plate in a density of 2×10⁵ cells per well and maintained at 37°C in 5% CO₂. The next day the medium was replaced with a fresh DMEM without antibiotics and cells were transfected by HA-mRNA using Lipofectamine 3000 reagent (Invitrogen) and Opti-Mem I Reduced Serum Medium (Gibco) in accordance with the manufacturer’s instructions. 24 h after transfection, cells were analyzed by immunocytochemical staining.

In vitro experiments were carried out with the use of the following continuous cell lines: osteoblast-like cells of human osteosarcoma MG-63 (CRL-1427^TM, American Type Cell Collection (ATCC)), human breast cancer (BC) cells MCF-7 (HTB-22^TM, ATCC), mouse macrophages RAW 264.7 (TIB-71, ATCC), and with the use of donor-derived primary cell culture of human bone marrow mesenchymal stem cells (MSC BM) of 3–8 passages. Obtaining an intraoperative sample of biological material from patient A was performed after voluntary informed consent of the patient and was approved by the Independent Ethics Council of the P.A. Herzen Moscow Cancer Research Institute. In vivo experiments were carried out with the use of the continuous cell line of murine mammary Ca-755 adenocarcinoma.
All manipulations with cell cultures were performed under standard aseptic conditions. The cells were cultured in complete growth medium (CGM) based on Dulbecco’s modified Eagles medium (DMEM) (PanEco, Moscow, Russia) supplemented with 10% fetal calf serum (HyClone, South Logan, UT, USA), 60.0 mg/mL L-glutamine (PanEco, Moscow, Russia), 50.0 µg/mL gentamicin (PanEco, Moscow, Russia), and 20.0 mM Hepes buffer (PanEco, Moscow, Russia) in humid air at 37°C and 5% CO₂. The medium was changed twice a week. Cell cultures in the preconfluent monolayer were used for experiments.

HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (PanEco, Moscow, Russia) supplied with 50 U/mL penicillin and 50 µg/mL streptomycin (PanEco, Moscow, Russia), 2 mM L-glutamine (PanEco, Moscow, Russia), and 10% fetal bovine serum (HyClone, Thermo Scientific, Waltham, USA) at 37 °C and 5% CO₂. For transient transfection, FuGENE HD reagent (Promega, Madison, USA) was used. A standard protocol was used to set up transfection; the DNA:reagent mixture was incubated for 20 min prior to the addition to the cells. Typically, 1 μg of DNA and 3 μL of the reagent were used. Transfection was performed in a serum-free OptiMEM (PanEco, Moscow, Russia). Immediately before imaging, DMEM was replaced with Hanks’ Buffer (PanEco, Moscow, Russia) supplemented with 20 mM HEPES (Sigma, Darmstadt, Germany).