Complete dmem medium

Manufactured by Merck Group

Sourced in United States

Complete DMEM medium is a cell culture medium that provides a balanced set of nutrients and growth factors to support the cultivation of a wide range of cell types in vitro. It is a formulation that includes essential amino acids, vitamins, inorganic salts, and other components required for cell proliferation and maintenance.

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9 protocols using complete dmem medium

Primary Cell Culture Protocol

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The cells were washed thoroughly with DPBS/Gentamycin thoroughly twice. These were centrifuged at 1500 rpm for 10 min. Supernatant was discarded. The pellet was re-suspended in complete DMEM medium (Sigma) and plated in a T25 flask and incubated at 37 °C under 5% CO₂. Media was changed every 3–4 days until the cells achieved 90% confluency. The cells were characterized through morphological evaluation and flow cytometry analysis.

Bansal H., Comella K., Leon J., Verma P., Agrawal D., Koka P, & Ichim T. (2017). Intra-articular injection in the knee of adipose derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. Journal of Translational Medicine, 15, 141.

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Osteogenic Differentiation of hUCMSCs

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hUCMSCs were seeded into six-well plates, at a density of 5 × 10⁵ per well. Upon attaining a 70–90% confluent, the cells were cultured in osteogenic induction medium [complete DMEM medium, dexamethasone (0.1 mM), ascorbic acid (50 ug/ml) and β-glycerolphosphate (10 mM) (all from Sigma-Aldrich)], while standard controls were cultured in complete medium.

Su C., Zheng X., He Y., Long L, & Chen W. (2021). Transcriptomic profiling and functional prediction reveal aberrant expression of circular RNAs during osteogenic differentiation in human umbilical cord mesenchymal stromal cells. Scientific Reports, 11, 19881.

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Culturing Murine Macrophage and Mammary Carcinoma Cells

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Murine macrophage Raw 264.7 cell line and 4T1 cells (murine mammary carcinoma cells) purchased from (ATCC® TIB-71™) were cultured in complete DMEM medium (Sigma, St. Louis, Mo, USA) supplemented with 10% fetal bovine serum (FBS) and 1% of penicillin and streptomycin and incubated at 37°C with 5% CO₂. When cell confluence reached around 80%, dead cells were washed away with PBS (Lonza) and live cells were detached by cell scraper. Cells were centrifuged and re-suspended with 8 ml of fresh DMEM medium. Cell counting was performed using BioRad TC20 cell counter.

Zambito G., Deng S., Haeck J., Gaspar N., Himmelreich U., Censi R., Löwik C., Di Martino P, & Mezzanotte L. (2021). Fluorinated PLGA-PEG-Mannose Nanoparticles for Tumor-Associated Macrophage Detection by Optical Imaging and MRI. Frontiers in Medicine, 8, 712367.

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Culturing HCT116 Colorectal Cancer Cells

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The human colorectal cancer cell line HCT116 (Public Health England) was grown in complete DMEM medium (Sigma-Aldrich, Dorset, UK) supplemented with 10% foetal bovine serum (FBS) (Invitrogen, Paisley, UK), 2 mM L-glutamine and 1% penicillin/streptomycin (both Sigma). Authentication of this cell line was performed by the service provider using the AmpFISTR Identifier Plus PCR amplification kit looking for the presence of <10 known loci for each cell line. Cells were incubated in a humidified atmosphere with 5% CO₂ in air at 37°C. When approximately 75% confluency was reached, cells were harvested with trypsin (Sigma-Aldrich) prior to washing and reseeding at a lower cell density. Only cells with a passage number <15 were used in experiments.

Gravett A.M., Dennis J.L., Dalgleish A.G., Copier J, & Liu W.M. (2020). The efficacy of chemotherapeutic drug combinations may be predicted by concordance of gene response to the single agents. Oncology Letters, 20(6), 321.

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Organotypic Co-culture Assay for Tubule Formation

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We performed an organotypic co-culture assay using FBs isolated from skin of EOS- and LSS-SSc, and HUVECs to investigate the effects of SSc-FBs on tubule formation in vitro, as previsouly described^{29 (link)}. Briefly, HC- or SSc-FBs were seeded at 2 × 10⁴ cells/well in a 24-well plate and grown until confluence for 7 days in complete DMEM medium. Subsequently, HUVECs were seeded on top of the FBs monolayer at 8.5 × 10³ cells/well in a 1:1 mixture of Human Large Vessel Endothelial Medium (TCS Cellworks) and complete DMEM medium (Sigma) supplemented with 10%FCS (Sigma) and rhEGFL7 100 ng/ml or VEGF 25 ng/ml, were added to the medium. Co-cultures were fixed for immunohistochemistry 5 days after HUVECs seeding. Fixation was carried out using ice-cold 70% ethanol for 30 min. ECs were successively labeled with mouse monoclonal anti-CD31 antibody^{45 (link)}.

Liakouli V., Cipriani P., Di Benedetto P., Panzera N., Ruscitti P., Pantano I., Berardicurti O., Carubbi F., Esteves F., Mavria G., Del Galdo F, & Giacomelli R. (2019). Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts. Scientific Reports, 9, 4589.

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Splenic T Cell Activation Assay

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Naïve spleens were excised from C3H/HeJ mice and processed to prepare a single cells suspension. The cells were seeded in U-bottom 96-well plates (Corning, United States) at a concentration of 5 × 10⁵ cells/well in complete DMEM medium (Sigma, United States) supplemented with 10% FBS (Atlanta for Biologicals, United States) 1% penicillin/streptomycin cocktail (Sigma, United States) and 50 µM mercaptoethanol (2-ME, Sigma, United States). Seeded cells were pretreated with either 50 µM RES or VEH for one hour before adding 1 μg/mL SEB to the culture. Activated cultures were incubated for 48 h at 37°C with 5% CO2 and the culture supernatants were collected for further analysis. Collected cultured cells were washed with phosphate buffer saline (PBS) twice before being suspended in FACS buffer. PE-conjugated anti-CD3 antibody (Biolegend, United States) was used to stain all harvested cells and then purified positively by using EasySep™ PE Positive Selection Kit II magnetic microbeads (STEMCELL, United States).

Alghetaa H., Mohammed A., Singh N., Wilson K., Cai G., Putluri N., Nagarkatti M, & Nagarkatti P. (2023). Resveratrol attenuates staphylococcal enterotoxin B-activated immune cell metabolism via upregulation of miR-100 and suppression of mTOR signaling pathway. Frontiers in Pharmacology, 14, 1106733.

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Isolation and Culture of Mouse Bone Marrow Macrophages

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Mouse BMMs were obtained from femurs of about 8-week-old BALB/c mice as stated previously^{36 (link)}. Briefly, bone marrow cavities were flushed with phosphate-buffered saline. After centrifugation at 500 g for 10 min, bone marrow cells were cultured for 7 days in DMEM complete medium supplemented with recombinant human CSF1 (10⁴ U/ml; Sigma-Aldrich) on a 10 cm Petri dish.

Usman M.W., Gao J., Zheng T., Rui C., Li T., Bian X., Cheng H., Liu P, & Luo F. (2018). Macrophages confer resistance to PI3K inhibitor GDC-0941 in breast cancer through the activation of NF-κB signaling. Cell Death & Disease, 9(8), 809.

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Isolation and Culture of Mouse Bone Marrow-Derived Dendritic Cells

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Bone marrow cells were isolated from the femur and tibia of C57BL/6 mice. Collected cells were treated with ACK buffer (Cambrex Bio Sciences, East Rutherford, NJ, usA) to lyse the red blood cells. Resulting cells were differentiated for 7 d at 1 × 10⁶ cells/ml in McCoy’s medium, supplemented with sodium bicarbonate (2.2 g/l), 10% FBS, antibiotics (100 µg/ml penicillin G and 50 µg/ml gentamycin), mouse recombinant GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) (Cell Sciences, Canton, MA, USA).^{33 (link)} At d 7, non-adherent cells were collected and positively selected for CD11c⁺ cells using CD11c Micro Beads (Miltenyi Biotec, San Diego, CA, USA), resulting in >98% CD11c⁺ population. Bone marrow-derived DCs (BMDCs) were cultured in DMEM complete medium supplemented with 10% FBS (Sigma-Aldrich) at 37°C with 5% CO₂. Experiments to obtain mouse-derived cells were approved by the University of Texas-Houston Health Science Center animal welfare committee (HSC-AWC-10-087 and HSC-AWC-11-158).

Hwang S.A., Kruzel M.L, & Actor J.K. (2014). Effects of CHO-expressed recombinant lactoferrins on mouse dendritic cell presentation and function. Innate immunity, 21(5), 553-561.

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Differentiation of Bone Marrow-Derived Macrophages

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Resulting cells were differentiated for 7 days at 1×10⁶ cells/mL in McCoy’s medium, supplemented with sodium bicarbonate (2.2 g/L), 10% FBS, antibiotics (100 μg/mL Penicillin G, and 50 μg/mL Gentamycin), mouse recombinant GM-CSF (10 ng/mL) (Cell Sciences, Canton, MA, USA). At day 7, non-adherent cells were washed away with 1×PBS. The resulting adherent cells (BM-Macs) were cultured in DMEM complete medium supplemented with 10% FBS (Sigma-Aldrich) at 37°C with 5% CO₂.

Hwang S.A., Kruzel M.L, & Actor J.K. (2015). CHO expressed recombinant human lactoferrin as an adjuvant for BCG. International journal of immunopathology and pharmacology, 28(4), 452-468.

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