Dmem cell culture

Manufactured by Thermo Fisher Scientific

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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium that provides the necessary nutrients and growth factors for the cultivation of a wide range of cell types, including adherent and suspension cells. It is a commonly used basal medium in cell culture applications.

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8 protocols using dmem cell culture

Oxidative Stress Measurement Protocol

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Haloperidol, carvacrol, phytohaemagglutinin (PHA), cytochalasin B, thiobarbituric acid, Tris-HCl, Tris ammonium, MgCl_2, Disodium hydrogen phosphate, TCA, sucrose, EDTA, sodium acetate, Triton X-100, and Giemsa stain were from Sigma; DMEM cell culture and phosphate buffered saline (PBS) were from Gibco. Phosphoric acid, methanol, acetic acid glacial, potassium chloride, n-Butanol, sodium chloride, DTNB, Na₂EDTA, sodium hydroxide, sodium lauroyl sarcosinate, DMSO, and normal melting point agarose were from Merck. The low melting point (LMP) agarose was from Cleaver Scientific.

Zamani E., Ahmadi Shad A., Fatemi H., Mahboubi S., Motavallian A, & Evazalipour M. (2022). Assessment of Protective Effects of Carvacrol on Haloperidol-Induced Oxidative Stress and Genotoxicity in Human Peripheral Blood Lymphocytes. Journal of Toxicology, 2022, 9565881.

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Cytotoxicity and Oxidative Stress Evaluation

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BSA (fraction V, minimum 98%) was purchased from Sigma (Steinheim, Germany). DMEM cell culture and fetal bovine serum (FBS) were purchased from Gibco (United States). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), penicillin, and streptomycin were all obtained from Sigma (United Kingdom), and dimethyl sulfoxide (DMSO) was purchased from Merck (Germany). 7-AAD, Annexin-V dyes, and annexin-V binding buffer were all obtained from BD Biosciences (San Jose, CA). All organic solvents used for TEM were received from VWR, Norway. 4,6-Diamidino-2-phenylindole (DAPI) was purchased from Invitrogen Life Technologies (Eugene, OR, USA). SNPs were synthesized according to the procedure reported by Eskandari et al.⁶².

Azizi M., Ghourchian H., Yazdian F., Bagherifam S., Bekhradnia S, & Nyström B. (2017). Anti-cancerous effect of albumin coated silver nanoparticles on MDA-MB 231 human breast cancer cell line. Scientific Reports, 7, 5178.

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Fractalkine-Induced Vascular Smooth Muscle Cell Activation

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HPASMCs were purchased from ScienCell Research Laboratories, Inc. (USA). DMEM cell culture and fetal bovine serum (FBS) were purchased from Gibco. TRQ (Cat. 1909120) was obtained from Shanghai Kaibao Pharmaceutical Co., Ltd (Shanghai, China). Recombinant Human Fractalkine/CX3CL1 (CX3CL1) and calcium fluorescent probe Fluo-3 AM and N-Acetyl-L-cysteine(cysteine) were purchased from Solarbio (Beijing, China). Reactive oxygen species detection kit and BCA protein concentration assay kit were supplied by Beyotime Co., Ltd. (Shanghai, China). Hydroxyl Free Radical Detection Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). SKF96365 was purchased from Selleck (Shanghai, China). Antibodies of TRPC1 and CX3CR1 were purchased from Santa Cruz. Antibodies of HIF-1α, NF-κBp65, and p-NF-κBp65 were purchased from Affinity. Desmin and SMMHC antibodies were purchased from ProteinTech Group, Inc. Human nuclear factor kB subunit p65 (NF-κBp65) ELISA kit was supplied by Jianglai bio. Co. Ltd. Fluor 488-phalloidin reagent was purchased from Abcam (ab176753).

Xiang Y., Zheng F., Zhang Q., Zhang R., Pan H., Pang Z., Dai S., Zhang Y., Wu Y., Yao L., Su M, & Lan L. (2022). Tanreqing Injection Regulates Cell Function of Hypoxia-Induced Human Pulmonary Artery Smooth Muscle Cells (HPASMCs) through TRPC1/CX3CL1 Signaling Pathway. Oxidative Medicine and Cellular Longevity, 2022, 3235102.

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Liver cell line culture protocol

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As the objective of our study, the normal liver cell lines (MIHA) and the HCC cell lines (MHCC-LM3, PRF-5, Huh-7, MHCC-97H, and Hep-G2) are originated from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Furthermore, they were maintained in a DMEM cell culture (Gibco, New York, United States) supplemented with 10% FBS (Gemini, West Sacramento, United States) in the incubator at 37°C with 5% CO2.

Shen B., Zhu W., Liu X, & Jiang J. (2022). NAP1L1 Functions as a Novel Prognostic Biomarker Associated With Macrophages and Promotes Tumor Progression by Influencing the Wnt/β-Catenin Pathway in Hepatocellular Carcinoma. Frontiers in Genetics, 13, 876253.

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Maintenance of Pancreatic Cancer Cell Lines

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The human pancreatic cancer cell lines, CFPAC-1 (RRID: CVCL_1119) and BxPC-3 (RRID: CVCL_0186), were obtained from Procell Life Science & Technology, Wuhan, China. CFPAC-1 and BxPC-3 were maintained in RPMI-1640 and Dulbecco's Modified Eagle Medium (DMEM) cell culture media (Gibco, Waltham, MA, USA), respectively. Both culture media were supplemented with 10% (v/v) fetal bovine serum (PAN, Adenbach, Bavaria, Germany), 50 units/ml Penicillin-Streptomycin (Gibco, USA). Cells were grown at 37°C in a humidified air and 5% CO₂ atmosphere. Both cell lines were characterized by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat markers within the last 3 years. All experiments were performed with mycoplasma-free cells.

Zhu Y., Peng X., Wang X., Ying P., Wang H., Li B., Li Y., Zhang M., Cai Y., Lu Z., Niu S., Yang N., Zhong R., Tian J., Chang J, & Miao X. (2022). Systematic analysis on expression quantitative trait loci identifies a novel regulatory variant in ring finger and WD repeat domain 3 associated with prognosis of pancreatic cancer. Chinese Medical Journal, 135(11), 1348-1357.

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Lentiviral Transduction of MSCs

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Human embryonic kidney 293T cells were cultured in 100mm plate for 24 hours before transfection. Three auxiliary plasmids (Rev, 2.5 μg; VSVG, 3 μg; pMD, 5 μg) were added when cell density reached to 60%-70%. 12 μg lentivirus vector was used for transfection. After plasmid and 293T cells incubated for 16 hours, the transfection effect was observed by fluorescence microscope (Nikon E400, Nikon Instruments, Inc., Tokyo, Japan). Virus was harvested by 0.45 μm filter after transfection for 24 hours. Filtered virus was concentrated in the PEG 6000 (GIBCO, Carlsbad, CA, USA) with final concentration of 8%-10% in 4℃ overnight, then centrifuged in 4℃ at 1500g for 45 minutes. The supernatant was discard, while the sediment cells were re-suspended with DMEM cell culture (GIBCO, Carlsbad, CA, USA), and then sterilized by 0.22 μm filter. The concentrated virus was added into MSCs with 1μl polybrene. Cell medium was changed within 8 to 12 hours after infection. The CPE was observed under fluorescence microscopy 48 hours after infection.

Wang Y., Peng D., Yang X., Huang P., Ye H., Hui Y., Wang X., Sun W., Wu H., Zhang S., Wang L., Sha H., Shang C., Dong H, & Hu Q. (2019). Study on Umbilical Cord-Matrix Stem Cells Transplantation for Treatment of Acute Traumatic Brain Injury in Rats. Turkish neurosurgery, 29(5).

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Evaluating Apoptosis Pathways in Cells

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3-(4 (link),5 (link)-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), anti-Cox IV, anti-Bcl-2, anti-Bax, anti-cleaved caspase 3, anti-cleaved caspase 9, anti-cleaved caspase 8, PARP, anti-phospho-Akt (Ser473), anti-Akt antibodies and anti-cytochrome c were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-PI3K p85α (Tyr467), and anti-PI3K p85α antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH was purchased from Bioworld Biotechnology. DAPI was obtained from Invitrogen (Grand Island, NY, USA). DMEM cell cultures and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). Taraxerol was obtained from Sigma (St. Louis, MO, USA).

Yao X., Lu B., Lü C., Bai Q., Yan D, & Xu H. (2017). Taraxerol Induces Cell Apoptosis through A Mitochondria-Mediated Pathway in HeLa Cells. Cell Journal (Yakhteh), 19(3), 512-519.

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Calycopterin Anticancer Mechanisms in Vitro

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Calycopterin, 5,4′-dihydroxy-3,6,7,8-tetramethoxyflavone was purified from D. kotschyi Boiss and the chemical structure was confirmed in our laboratory as reported previously [20 (link)]. MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide), dimethylsulfoximine (DMSO), and propidium iodide (PI) were purchased from Sigma (St. Louis, MO, USA). Anti-cell division cycle 25 homolog C (cdc25c), anticyclin B1, anti-Bcl-, anti-Bax, anti-pro-caspase 3, anti-pro-caspase 9, anti-poly ADP-ribose polymerase (PARP), anti-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-c-Jun N-terminal kinase (JNK), anti-p38 MAPK, anti-Akt, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-JNK (Thr183/Tyr185), anti-phospho-p38 MAPK (Thr180/Try182), anti-phospho-Akt (Ser473), and anti-phospho-phosphatidylinositol-3-kinase (PI3K) (Tyr458/Tyr199) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Benzyloxycarbonyl–Val–Ala–Asp–fluoromethyl ketone (z-VAD-fmk), LY294002, U0126, SP600125, and SB203580 were purchased from Promega (Madison, WI, USA). DMEM cell cultures, fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). All other reagents were commercial products of the highest available purity grade.

Esmaeili M.A., Farimani M.M, & Kiaei M. (2014). Anticancer effect of calycopterin via PI3K/Akt and MAPK signaling pathways, ROS-mediated pathway and mitochondrial dysfunction in hepatoblastoma cancer (HepG2) cells. Molecular and cellular biochemistry, 397(1-2), 17-31.

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