Dulbecco s modified essential medium

Fetal livers were obtained by dissection and collected in 1 mL of MSC media consisting of Dulbecco’s modified essential medium low glucose, 10% fetal bovine serum, 1% L-glutamine (all Life Technologies, CA, USA), and 1% Antibiotic-Antimycotic (100×, Gibco, Sigma-Aldrich, Missouri, USA), as previously described [18 (link)]. The aMSCs were isolated from 10 to 20 mL bone marrow aspirates and expanded as previously described [19 (link)], using the same MSC media as used for fMSCs. For the experiments in this study, the fetal and adult MSCs were in Passage (P) 5–8. After isolation, the MSCs were plated at 4000 cells/cm².

Huh7 and HepG2 cells were obtained from the American Type Culture Collection (Rockville, Maryland, USA). Both cell lines were cultured in Dulbecco's modified essential medium (Life Technologies, Carlsbad, California, USA) supplemented with 10% heat‐inactivated fetal calf serum (Life Technologies) and 100 U/mL penicillin and 100 μg/mL streptomycin sulfate (Life Technologies) in a humidified incubator with 5 % CO₂ at 37°C.
For experimental conditions, Interferon (IFN)‐alpha (IFN‐α), IFN‐gamma (IFN‐γ), N'‐hydroxy‐N‐phenyloctanediamide (SAHA) as well as ionomycin and phorbol 12‐myristate 13‐acetate (PMA) were obtained from Sigma‐Aldrich (St. Louis, Missouri, USA) and dissolved as described by manufacturer's instructions.

U87 cells grew (37 °C, 5 % CO₂) in Dulbecco’s modified essential medium (Life Technologies) supplemented with 10 % heat-inactivated fetal calf serum (PAA), 100 U/ml penicillin (PAA), 100 μg/ml streptomycin (PAA), and 1 % NEAA (Life Technologies).

MDA‐MB‐468 and MDA‐MB‐231 cells were maintained in Dulbecco's modified essential medium (Life Technologies, Carlsbad, CA, USA) supplemented with 1% penicillin‐streptomycin solution (Life Technologies) and 10% fetal bovine serum (HyClone, Logan, UT, USA). T47D cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin‐streptomycin solution and 10% fetal bovine serum. MCF10A cells were maintained in DMEM/F12 media with 10% horse serum, 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 500 ng/mL hydrocortisone, 0.01 mg/mL insulin and 1% penicillin‐streptomycin solution. In the current study, all cell lines used were all origin from ATCC and used within 6 months after authenticated via the short tandem repeat (STR) typing.

Three cancer cell types were studied in the present study: highly radioresistant U87 glioblastoma cancer cells, radioresistant SkBr3 breast cancer cells and relatively less radioresistant HeLa cervix cancer cells. U87 and HeLa cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). SkBr3 was commercially available and used for several SMLM studies in our laboratory. U87 and HeLa cells were grown in Dulbecco’s modified essential medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin (PAA), 100 μg/mL streptomycin (PAA), and 1% NEAA (Thermo Fisher Scientific, Waltham, MA, USA). Cell cultures were kept in T-25 cell flasks at 37 °C in a humidified atmosphere with 5% CO₂.
For the experiments with SMLM, SkBr3 cells were prepared as described in detail elsewhere [72 (link)]. SkBr3 cells were grown in McCoy’s 5A cell medium, containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultivated and maintained at 37 °C in a humidified atmosphere at 95% air/5% CO₂. Then, the cells were trypsinized and transferred to coverslips, put into six-well plates and further cultivated (about three passages, i.e., about 38 h) until 80% confluence.

Human mesenchymal stem cells (hMSCs) were purchased from a commercial source (Lonza Biologics, Slough, UK) and expanded up to passage number 3. These cells were derived from human bone marrow of a Caucasian female aged 29. hMSCs were cultured in Alpha Minimum Essential medium (Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, East Grinstead, UK). Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium supplemented with EGM2 SingleQuot Kit supplement and growth factors. HeLa cells were cultured in Dulbecco’s modified essential medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum and 1% Antibiotic-Antimycotic. Cells were cultured at 37°C and 95% air/ 5% CO₂ with the medium being changed every other day.
For fixed cell experiments, media was aspirated and samples were washed with phosphate buffered saline (PBS, Invitrogen) before fixing with 3.7% paraformaldehyde solution after 6 h of culture on nanoneedle arrays (NNAs). For visualization under an upright microscope, cells were incubated with 0.5μg/ml of wheat germ agglutinin (WGA) Alexafluor 488 Conjugate (Thermo Fisher Scientific) for 10 min and washed in PBS. For live experiments, cells were stained with WGA and then supplemented with Lebovitz-15 media at 37°C (Thermo Fisher Scientific).