Dmem high glucose medium

Human MV3 melanoma cells were cultivated in RPMI 1640 medium (PAN Biotech, Aidenbach, Germany) containing 10% (v/v) fetal calf serum (FCS) (Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (PAN Biotech). EA.hy926 endothelial cells were cultured in Dulbecco’s modified Eagle’s Medium low glucose (DMEM low glucose) (Sigma Aldrich) with 10% FCS, 100 U/mL penicillin and 10 μg/mL streptomycin. MDA-MB-231 breast cancer cells were maintained in DMEM high glucose medium (PAN Biotech) and supplemented with 10% FCS, 1% l-glutamine (PAN Biotech), 100 U/mL penicillin and 100 μg/mL streptomycin. PC-3 prostate cancer cells were cultivated in RPMI 1640 medium containing 10% (v/v) fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1% l-glutamine and 1% sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA). All cells were incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂. For subcultivation, MV3, PC-3, MDA-MB-231 and EA.hy926 cells were detached at a confluency of about 90% with trypsin/EDTA (5 g/L trypsin; 0.2 g/L EDTA × tetra sodium, Sigma Aldrich) for 5 min at 37 °C. Test for absence of mycoplasms were performed routinely every month.

FAP and MuSC isolation from muscle was performed as described in^{25 (link)}. Briefly, TA muscle was isolated and roughly minced with a small scissor in high-glucose DMEM medium (PAN Biotech) containing 10% fetal bovine serum (PAN biotech), 1% Penicillin Steptomycin (P/S) solution (PAN Biotech, 10.000 U/ml) and 2,5 mg/ml Collagenase A (Roche) for 45 min at 37 °C with gentle shaking. Muscle lysates were further digested with 2 IU/ml of Dispase II (Sigma Aldrich) diluted in minimum amount of PBS for 30 min. To stop the enzymatic digestion, 10% FCS DMEM was added to the sample and subsequently the sample was passed ten times through a 20 G syringe needle. Cells were filtered first through a 70 µm and then through a 40 µm cell strainer (Fischer Scientific) and collected by centrifugation at 400xg for 10 min. Cells were resuspended in filtered “FACS buffer” containing 1% BSA and 2 mM EDTA, labeled with the following antibodies: anti-CD45-APC, anti-Cd31-APC, anti-TER119-APC, anti-α7-integrin-PE, and anti-Ly6A/e-APC-Cy7 (Table S4) for 30 min on ice and washed three times with FACS buffer prior to sorting. Propidium iodide was used as a viability dye. Cell sorting and analysis was performed on a BD FACSAriaII SORP (BD Biosciences). Single-stained and fluorescence minus-one controls were used for setting the sorting gates. Data were collected using the BD FACSDiva software version 8.0.1.

The antiproliferative activity of bran extracts was tested on four cell lines—three of them are human cancer cells and one is the normal cell line. MCF-7 and T47D are human epithelial breast cancer cell lines, while MDA-MB-231 is human hormone-independent breast cancer cells. EMT6/P and fibroblast are the mouse epithelial breast cancer cell line and human skin fibroblast cells, respectively. To achieve a successful cell culture, many factors were followed such as using completed medium and incubating cells in 5% CO₂ at 37 °C. Based on the type of the cells, the type of culturing medium varied. For MCF-7 and T47D, complete RPMI 1640 medium (PAN-biotech, Aidenbach, Germany) was used, while complete MEM medium (PAN-biotech, Aidenbach, Germany) was used for culturing EMT6/P. High-glucose DMEM medium (PAN-biotech, Aidenbach, Germany) was utilized for MDA-MB-231 and fibroblast cell lines. In this context, a complete culture medium was prepared through adding the following supplements with the required percentage of each type of tissue culture medium. The supplements are: 1% L-glutamine (Sigma, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, Brough, UK), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA), 0.1% non-essential amino acids (Sigma, St. Louis, MO, USA) and 0.1% gentamycin solution (Sigma, St. Louis, MO, USA).

In this study, four adherent cell lines were used: three from different cancer types and one from a regular fibroblast. The cancerous cells were human breast carcinoma (MDA-MB-231, ATCC code: HTB-26), human prostatic carcinoma (PC-3, ATCC code: CRL-1435), and human lung cancer epithelial cells (A549, ATCC code: CCL-185), while the fibroblasts in the control group were human fetal lung (MRC-5, ATCC code: CCL-171) cells. All cells were checked every few weeks for various sources of contamination.
MDA-MB-213, MRC-5, and PC-3 cell lines were cultured in T75 flasks with high glucose DMEM medium (Pan Biotech, Aidenbach, Germany), and A549 cells were cultured with RPMI-1640 (Pan Biotech) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA), 1% penicillin-streptomycin (Gibco), and 1% l-glutamine (Gibco) at 37 °C with 5% CO₂. All the cells were removed from the flask with Trypsin/EDTA 0.25% (Gibco) when they reached 80% density.