MDA-MB 231 cells were obtained from ATCC (Manassas, VA, USA), whereas SUM159 cells were kindly provided by Dr. W.T. Khaled (University of Cambridge, UK). Cells were used less than 6 months after thawing and routinely tested and authenticated according to the ATCC suggestions. MDA-MB 231, MDA-MB 231 PTK2 WT and MDA-MB 231 PTK2 KO cells (see below for technical details) were maintained in DMEM/F12 (Dulbecco’s modified Eagle’s medium) (Life Technologies, Milan, Italy) with phenol red, supplemented with 5% FBS and 100 μg/mL of penicillin/streptomycin. SUM159, SUM159 PTK2 WT and SUM159 PTK2 KO cells (see below for technical details) were maintained in DMEM/F12 (Dulbecco’s modified Eagle’s medium) (Life Technologies, Milan, Italy) with phenol red, supplemented with 1 μg/mL of insulin, 1 μg/mL of hydrocortisone, 5% FBS and 100 μg/mL of penicillin/streptomycin. Cells were grown in a 37 °C incubator with 5% CO2. Cells to be processed for immunoblot and RT-PCR assays were switched to medium without serum and phenol red 24h before treatments.
Dulbecco s modified eagle s medium dmem f12
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy, China, Japan
Dulbecco's modified Eagle's medium (DMEM)/F12 is a cell culture medium formulation commonly used for the in vitro cultivation of various cell types. It provides a balanced salt solution and essential nutrients required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (49 mentions)
Trypsin, by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Collagenase 2, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Poly d lysine, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
97 protocols using dulbecco s modified eagle s medium dmem f12
1
Cell Culture and Maintenance Protocol
2
TNBC Cell Line Maintenance and Handling
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TNBC cell lines MDA-MB 231 were obtained from ATCC (Manassas, VA, USA). TNBC cell lines SUM159 were kindly provided by Dr. W.T. Khaled, University of Cambridge, UK. Cells were used less than 6 months after resuscitation and routinely tested and authenticated according to the ATCC suggestions. MDA-MB 231 cells were maintained in DMEM/F12 (Dulbecco’s modified Eagle’s medium) (Life Technologies, Milan, Italy) with phenol red, supplemented with 5% FBS and 100 μg/ml of penicillin/streptomycin. SUM159 cells were maintained in DMEM/F12 (Dulbecco’s modified Eagle’s medium) (Life Technologies, Milan, Italy) with phenol red, supplemented with 1 μg/ml of insulin, 1 μg/ml of hydrocortisone, 5% FBS and 100 μg/ml of penicillin/streptomycin. MDA-MB 231 and SUM159 cells were grown in a 37 °C incubator with 5% CO2. Cells to be processed for immunoblot and RT-PCR assays were switched to medium without serum and phenol red the day before treatments.
3
3D Lung Organoid Culture Protocol
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Lung epithelial cells (Ter119−/Cd31−/Cd45−/Epcam+) isolated from 7-10-week-old Cracd WT mice or Cracd KO were cultured with lung stromal cells in a 3D organoid air-liquid interface, as described previously24 (link),45 (link). In brief, freshly sorted lung epithelial cells were resuspended in 3D organoid media (Dulbecco’s modified Eagle’s medium [DMEM]/F12 [Gibco, USA]), 10% FBS [Thermo Fisher Scientific], 1′ penicillin-streptomycin-glutamine [Thermo Fisher Scientific], and 1′ insulin-transferrin-selenium [Thermo Fisher Scientific.]) and mixed with LuECs at a ratio of 1:1. Cells containing 3D media were mixed with growth factor-reduced Matrigel (BD Biosciences) at a ratio of 1:1. The 100 ml of mixtures containing lung epithelial cells (5 X 103) and LuECs (5 X 104) were placed in the transwell insert (0.4-mm pore, Corning, Lowell, MA). After incubation for 30 mins at 37°C in an incubator, 500 ml of 3D media was placed in the bottom chamber to generate the liquid-air interface. Media were exchanged every other day.
4
Chondrocyte Isolation and Culture Protocol
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Chitosan (molecular weight, 5 kDa; deacetylation degree, 90%), and Chitosanase were purchased from Sigma-Aldrich (USA). Sodium hyaluronate (molecular weight, 35 kDa) was purchased from Freda Biochem Co., Ltd. (Shandong, China). Deoxyribonuclease I (DNase I), SYBR Prime Ex Taq II were obtained from Invitrogen (USA). The streptavidin-biotin-peroxidase complex (SABC) kit and the primary antibodies against matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were purchased from Boster (Wuhan, China). Collagenase II, trypsinase, antibiotics, Dulbecco’s modified Eagle’s medium (DMEM)/F12, and fetal bovine serum (FBS) were obtained from Gibco. All reagents used in this study were of analytical grade.
5
Oxidative Stress and Apoptosis Assay
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Fluorescent probe 2,7-dichlorofluorescein diace-tate (DCF-DA), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), Triton X-100, FBS and Rhodamine-123 were purchased from Sigma(St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM-F12) was purchased from Gibco (Gibco, Grand Island, NY, USA). Caspase-3 Detection Kit was provided from Sigma. Super Oxide Dismutase Assay Kits were purchased from Cayman (Ann Arbor, MI, USA). LiChroprep® RP-18 (15-25 μm) were purchased from Merck (Darmstadt, Germany) and all the solvents used for extraction from Scharlau and Caledon (Sentmenate, Spain).
6
Evodiamine Modulates Chondrocyte Inflammation
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In this study, evodiamine (purity >98%), dimethylsulfoxide (DMSO), and type II collagenase were obtained from Solarbio (Beijing, China). Mouse IL-1β was purchased from Novoprotein (Suzhou, China). The following primary antibodies were utilized in the present study: mouse anti-collagen II, mouse anti-ADAMTS-5, mouse anti-COX-2, mouse anti-IκB, and mouse anti-GAPDH were purchased from Affinity Biosciences (Beijing, China). Mouse anti-iNOS, mouse anti-MMP-13, and mouse anti-Laminb1 were acquired from Proteintech (Wuhan, China). Primary antibodies to mouse anti-p65 were obtained from Cell Signaling Technology (Boston, MA, United States). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM)/F12 were acquired from Gibco (Grand Island, United States). And all ELISA kits were acquired from Cusabio (Wuhan, China).
7
Immortalization of Human Spermatogonial Stem Cells
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By transfecting Large T antigen into G protein-coupled receptor 125 (GPR125)-positive human undifferentiated spermatogonia, immortalized human SSC lines were established[18 (link)]. Immortalized human SSCs maintained many properties of their primary cells and expressed many markers of primary SSCs including GFRA1, RET, and promyelocytic leukemia zinc finger (PLZF). They did not express testicular endosomal cell markers such as SRY-box transcription factor 9[15 (link)]. The immortalized human SSCs were grown at 34 °C with 50 mL/L CO2 in an incubator, and the culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY, United States) supplemented with 100 mL/L fetal bovine serum (FBS; Gibco). The cells were subcultured every 2 d or 3 d (0.5 g/L trypsin and 0.53 mmol/L Ethylenediaminetetraacetic acid (EDTA); Invitrogen, Carlsbad, CA, United States).
8
Isolation and Culture of UC-MSCs
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UC-MSCs were obtained and isolated as previously reported (19 (link),20 (link)). Briefly, umbilical cords were collected from term infants immediately after birth. The cords were rinsed twice in phosphate-buffered saline (PBS) with 100 units/mL penicillin and streptomycin, and the cord blood vessels were carefully removed. Wharton’s jelly was cut into 1–2 mm3 pieces and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 containing 10% fetal bovine serum (FBS) (all from Gibco, Life Technologies). After culture for 2 days, the medium containing non-adherent cells was discarded, and the medium was replaced twice a week thereafter. MSCs between passages 4 to 6 were used for subsequent experiments.
9
Cell Viability Assay with Withaferin A
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PBS (pH = 7.2, 1x), 0.25% trypsin–EDTA (1x), Dulbecco's Modified Eagle's Medium DMEM/F-12 (1x), 0.4% trypan blue, and antibiotic/antimycotic solution (100×) were obtained from Gibco, Life Technologies; whereas fetal bovine serum (FBS) and MTT were obtained from Himedia. Doxorubicin hydrochloride solution was obtained from Sigma Chemical Co. (St. Louis, MO, USA) and dimethyl sulfoxide (DMSO) from Calbiochem. Withaferin A standard was from Natural Remedies Ltd. Veerasandra, Bangalore-100. All HPLC grade reagents were used in HPLC. All other chemicals were of analytical grade.
10
In-vitro Neurodegeneration Assay
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All procedures were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals. MPP+, recombinant α-synuclein, cobalt protoporphyrin IX (CoPPIX), zinc protoporphyrin (ZnPP), deferoxamine (DFO), methylene blue (MB) and primary antibodies against microtubule-associated protein 2 (MAP2) and GFAP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary HO-1 antibody was purchased from Stressgen Bioreagents (Ann Arbor, MI, USA). Primary mitochondria ferritin (MtFt) was purchased from Abcam (Cambridge, UK). Primary cytochrome c oxidase subunit IV (COX4) was purchased from Takara Biomedical Technology (Beijing, China). Rhodamine 123 was purchased from Invitrogen (Madrid, Spain). Caspase-3 Kit was from BD Biosciences, Dulbecco’s modified Eagle’s medium (DMEM)/F12, B27 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). All other chemicals and reagents were of the highest grade available and were obtained from local commercial sources.
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