Advanced dmem

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Advanced DMEM is a cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides a balanced mixture of nutrients, vitamins, and other essential components required for optimal cell proliferation and survival in vitro.

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Advanced DMEM/F12 (505 citations) Advanced DMEM/F12 medium (111 citations) Advanced DMEM medium (24 citations) Advanced DMEM-F12 media (22 citations) Advanced® DMEM/F-12 culture medium (7 citations) Advanced Dulbecco’s modified Eagle’s medium (DMEM), (5 citations) Advanced DMEM/F12 + GlutaMAX™ (3 citations) Advanced DMEM/F12 with N2 (3 citations) Advanced DMEM media (3 citations) SILAC Advanced DMEM/F-12 Flex media (2 citations)

195 protocols using advanced dmem

Isolation of Extracellular Vesicles from Cell Culture

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The cells were washed with phosphate‐buffered saline without calcium chloride and magnesium chloride (PBS−), and the culture medium was replaced with advanced DMEM (Thermo Fisher Scientific) for DU145 and MG63 cells, advanced RPMI 1640 medium (Thermo Fisher Scientific) for 22Rv1 cells, and advanced DMEM (Thermo Fisher Scientific)/advanced DMEM/F12 (Thermo Fisher Scientific) (3 : 2) for C4‐2 and C4‐2B cells. Each advanced medium contained 1% AA and 2 mm l‐glutamine. EVs from the CM of cells were isolated by a differential ultracentrifugation protocol, as we previously reported [16 (link)]. Briefly, the CM was centrifuged at 2000 g for 10 min to remove contaminating cells. The resulting supernatants were then transferred to fresh tubes and filtered through a 0.22 μm filter (Millipore, Burlington, MA, USA). The filtered CM was centrifuged for 70 min at 110 000 g to pellet the enriched EVs (Beckman Coulter, Rea, CA, USA). The pellets were washed with 11 mL of PBS and ultracentrifuged at 110 000 g for another 70 min. The EV pellets were stored in the refrigerator at 4 °C until use. The fraction containing the EVs was measured for its protein content using the Quant‐iTTM Protein Assay with Qubit2.0 Fluorometer (Invitrogen).

Ito K., Yamamoto T., Hayashi Y., Sato S., Nakayama J., Urabe F., Shimasaki T., Nakamura E., Matui Y., Fujimoto H., Kimura T., Egawa S., Ochiya T, & Yamamoto Y. (2023). Osteoblast‐derived extracellular vesicles exert osteoblastic and tumor‐suppressive functions via SERPINA3 and LCN2 in prostate cancer. Molecular Oncology, 17(10), 2147-2167.

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Prostate and Pheochromocytoma Cell Culture and Lysate Preparation

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Human prostate cancer cell line PC-3 cells (obtained from ATCC, Manassas, USA; ATCC Number: CRL-1435) were cultured in F-12 (Gibco) and Advanced DMEM (Gibco) (1:1) with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine. Rat pheochromocytoma PC-12 cells (obtained from ATCC, Manassas, USA; ATCC Number: CRL-1721) were cultured in Advanced DMEM (Gibco) with 5% FBS, 10% heat-inactivated horse serum, 1% penicillin/streptomycin and 1% L-glutamine.
For testing the enzyme activity in cell lysates, cells were treated with 10 μM of compounds. After 24 hours lysates were prepared in 50 mM Na acetate buffer pH 5.5 with 1 mM EDTA, 100 mM NaCl and 0.25% Triton X-100. Total protein concentration was determined by DC Protein Assay (Bio Rad) according to the instructions.

Fonović U.P., Mitrović A., Knez D., Jakoš T., Pišlar A., Brus B., Doljak B., Stojan J., Žakelj S., Trontelj J., Gobec S, & Kos J. (2017). Identification and characterization of the novel reversible and selective cathepsin X inhibitors. Scientific Reports, 7, 11459.

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Isolation and Culture of Astrocytes from APOE Mice

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Primary astrocytes were isolated from postnatal day 0–4 pups of mice homozygous for E3 or Ε4. The brain was surgically excised and meninges were removed from cortical tissue in cold DMEM. Tissue from pups of the same genotype was pooled and coarsely chopped to encourage suspension. Tissue homogenates were incubated in serum free DMEM with 0.25% trypsin and DNAse for 30 min with gentle shaking. Cell suspension was then filtered through 40 μm strainer and spun for 5 min at 1100 x g. Suspended primary cells were then plated in a poly-lysine coated plate and allowed to grow to confluence in Advanced DMEM (Gibco) with 10% FBS. Immortalized astrocytes were derived from targeted replacement mice expressing human APOE alleles (kind gift from Dr. David Holtzman). These immortalized cell lines secrete human ApoE in HDL-like particles at equivalent levels to primary astrocytes from targeted replacement APOE knock-in mice and have been relied upon for studies of APOE’s role in astrocyte metabolism by several groups [39 (link)–41 (link)]. Cells were maintained in Advanced DMEM (Gibco) supplemented with 1 mM sodium pyruvate, 1X Geneticin, and 10% fetal bovine serum unless otherwise noted.

Farmer B.C., Williams H.C., Devanney N.A., Piron M.A., Nation G.K., Carter D.J., Walsh A.E., Khanal R., Young L.E., Kluemper J.C., Hernandez G., Allenger E.J., Mooney R., Golden L.R., Smith C.T., Brandon J.A., Gupta V.A., Kern P.A., Gentry M.S., Morganti J.M., Sun R.C, & Johnson L.A. (2021). APOΕ4 lowers energy expenditure in females and impairs glucose oxidation by increasing flux through aerobic glycolysis. Molecular Neurodegeneration, 16, 62.

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Measurement of Calcium Fluxes in Activated T Cells

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For the measurement of calcium fluxes, preactivated AND T cells were loaded with the calcium sensitive dye fura-2 AM (Life Technologies). AND T cells were pelleted at 700 rpm, washed once with 10 mL of serum-free Advanced DMEM (Life Technologies), and resuspended in 1 mL of room temperature serum-free Advanced DMEM containing 2 μM Fura-2. Cells were then incubated at room temperature for 25 min in the dark. After this, 5 mL of pre-warmed ADMEM containing 2.5% serum was added to the cells, and the cells were incubated for an additional 1 h. at 37 °C. The cells were then pelleted at 700 rpm and used directly for imaging. The cells were imaged with a 40X, 1.35 NA objective and the fluorescence emission recorded for 340 and 380 nm excitation. IRM and ICAM-1 images were also collected so that cells adhering to the bilayer could be identified. As a control to determine the maximum calcium flux, the cells were subjected at the end of the experiment to treatment with 2 μg/mL ionomycin in HBS/BSA containing 2 mM MgCl₂ and 20 mM CaCl₂. Finally, as a control to measure the baseline Fura-2 fluorescence emission, the cells were subjected to treatment with 2 mM EGTA in calcium-free HBS/BSA containing 3 mM MgCl₂.

Crites T.J., Padhan K., Muller J., Krogsgaard M., Gudla P.R., Lockett S.J, & Varma R. (2014). TCR Microclusters Preexist and Contain Molecules Necessary for TCR Signal Transduction. Journal of immunology (Baltimore, Md. : 1950), 193(1), 56-67.

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Extraction and Characterization of Bioactive Wood Compounds

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Commercially available wood extracts rich in HT were obtained from Tanin d.d. Sevnica (Sevnica, Slovenia). Tanex and Farmatan originate from sweet chestnut wood (Castanea sativa Mill.), and Contan from oak (Quercus patraea). Gallic acid (Sigma, Steinheim, Germany) was used as a reference compound. All wood extracts and Gallic acid were in powder form before use. The chemical compositions of the wood extracts are summarized in Table 1. Suitable quantities of wood extracts and Gallic acid were resuspended in Advanced DMEM supplemented with 2 mM L-glutamine (Life Technologies, Bleiswijk, The Netherlands), vigorously vortexed for 30 min at room temperature to dissolve the powder, and centrifuged at 10,000× g for 10 min. After centrifugation, supernatants were collected and further diluted to the desired concentrations in Advanced DMEM supplemented with 2 mM L-glutamine (Life Technologies, Bleiswijk, The Netherlands).

Brus M., Frangež R., Gorenjak M., Kotnik P., Knez Ž, & Škorjanc D. (2021). Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model. Molecules, 26(2), 345.

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Purification of Lipidated apoE Particles

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Lipidated apoE isoform particles were purified from culture media of human apoE3 or apoE4 overexpressing immortalized astrocytes using an affinity column, as we described previously [53 (link)]. Briefly, astrocytes were cultured in advanced DMEM (Invitrogen) with 10% FBS. After 90–95% confluency, cells were washed by PBS and further incubated in advanced DMEM with N-2 Supplement (Invitrogen) and 3 mM 25-hydroxycholesterol (Sigma) for 3 days. Collected culture media were applied onto mouse monoclonal antibody against a human apoE (WU E-4) column. Lipidated apoE particles were eluted from the column with 3 M sodium thiocyanate, concentrated using Apollo centrifugal quantitative concentrators (QMWL: 150 kDa, Orbital Biosciences), and dialyzed against PBS.

Ma Q., Zhao Z., Sagare A.P., Wu Y., Wang M., Owens N.C., Verghese P.B., Herz J., Holtzman D.M, & Zlokovic B.V. (2018). Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism. Molecular Neurodegeneration, 13, 57.

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Isolation and Culture of Human Immune Cells

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Peripheral blood mononuclear cells (PBMC) were obtained by density-gradient centrifugation and red blood cell lysis from peripheral blood of healthy donors in agreement with institutional consent and collection guidelines (Institute for Transfusion Medicine and Haemotherapy, University Hospital Wurzburg and Wurzburg University Ethic Committee, No. 141/17-sc). The human cell lines THP-1 (acute myeloid leukemia, ATCC, No. TIB-202 and DSMZ, No. ACC-16), HT1080 (sarcoma, ATCC, No. CCL-121), U266 (multiple myeloma, ATCC, No. TIB-196), Jurkat (T cell leukemia, DSMZ, No. ACC-282), KMS-12-BM (multiple myeloma, DSMZ, No. ACC-551), Raji (Burkitt lymphoma, ATCC, No. CCL-86), and MDA-MB-231 (ATCC, No. HTB-26) were grown in advanced RPMI-1640 or advanced DMEM supplemented with 200 μM l-glutamine, 10% FBS, penicillin (200 U/mL), and streptomycin (200 μg/mL) (Thermo Fisher Scientific, MA, USA). We routinely tested cell lines for absence of mycoplasma infection. Authentication of the cells was performed by the provider.

Banaszek A., Bumm T.G., Nowotny B., Geis M., Jacob K., Wölfl M., Trebing J., Kucka K., Kouhestani D., Gogishvili T., Krenz B., Lutz J., Rasche L., Hönemann D., Neuweiler H., Heiby J.C., Bargou R.C., Wajant H., Einsele H., Riethmüller G, & Stuhler G. (2019). On-target restoration of a split T cell-engaging antibody for precision immunotherapy. Nature Communications, 10, 5387.

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Glutamine Assay in Cell Culture

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A total of 5 × 10⁴ cells/well were cultured in 96-well plates containing advanced DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 2 mM glutamine, and then transferred to a CO₂ incubator set at 37 °C, 100% humidity, and 5% CO2 at specified times. The supernatant media were collected to measure the remaining glutamine using a glutamine assay kit (Abcam, Cambridge, MA, USA) following the manufacturer's instructions.

Zhao H., Jiang Y., Lin F., Zhong M., Tan J., Zhou Y., Liu L., Li G., Deng M, & Xu B. (2022). Chidamide and apatinib are therapeutically synergistic in acute myeloid leukemia stem and progenitor cells. Experimental Hematology & Oncology, 11, 29.

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Intestinal Organoid Establishment from Crypts

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Isolation of the crypts and the subsequent establishment of intestinal organoids were performed as previously described [25 (link), 26 (link)]. Briefly, crypts were collected by rigorously shaking biopsy specimens in 15 mM EDTA. Isolated crypts were embedded in 30 μl of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at a density of 20–30 crypts per well and placed in 24-well culture dishes. The crypts were maintained in standard culture medium (WENR medium) consisting of basal medium (Advanced-DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with recombinant human R-spondin-1 (1 μg/ml, R&D Systems, Minneapolis, MN, USA), recombinant human Wnt-3a (300 ng/ml, R&D Systems, Minneapolis, MN, USA), recombinant human Noggin (100 ng/ml, R&D Systems, Minneapolis, MN, USA), recombinant human EGF (50 ng/ml, PeproTech, Rocky Hill, NJ, USA), A83-01 (500 nM, Sigma-Aldrich Japan Tokyo, Japan) and SB202190 (10 μM, Sigma-Aldrich Japan, Tokyo, Japan). Y-27632 (10 μM, Sigma-Aldrich Japan, Tokyo, Japan) was added to the culture medium for the initial 3 days. Phase-contrast views of the established organoids were captured by a CCD-equipped microscope (BZ-X700, Keyence, Osaka, Japan).

Suzuki K., Murano T., Shimizu H., Ito G., Nakata T., Fujii S., Ishibashi F., Kawamoto A., Anzai S., Kuno R., Kuwabara K., Takahashi J., Hama M., Nagata S., Hiraguri Y., Takenaka K., Yui S., Tsuchiya K., Nakamura T., Ohtsuka K., Watanabe M, & Okamoto R. (2018). Single cell analysis of Crohn’s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. Journal of Gastroenterology, 53(9), 1035-1047.

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Imaging Translating RNA in Intestinal Organoids

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To enable imaging of single translating vRNAs in intestinal organoid cells, organoid cells were cultured in 2D. 96-well glass-bottom plates (Matriplates, Brooks Life Science Systems) were coated for 30 minutes at 37°C with 100 μL coating mix (5% Basement Membrane Extract (Type 2, Cultrex reduced growth factor, R&D systems) dissolved in Advanced DMEM (Thermofisher Scientific)). The coating mix was removed and the wells were dried for 15 minutes at 37°C. Organoids grown in 3D were dissociated into single cells by incubating the organoids for 5 minutes with TrypLE (TryplE Express; Life Technologies). Approximately 5000 cells were plated per well in expansion medium (see Sato et al., 2011 (link)). Virus incubation was performed as described in the section ‘Cell culture for imaging’. Expansion medium was replaced with imaging medium (pre-warmed CO₂-independent Leibovitz’s-15 medium (GIBCO) containing 5% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (GIBCO)) 15-30 minutes before the start of live-cell imaging.

Boersma S., Rabouw H.H., Bruurs L.J., Pavlovič T., van Vliet A.L., Beumer J., Clevers H., van Kuppeveld F.J, & Tanenbaum M.E. (2020). Translation and Replication Dynamics of Single RNA Viruses. Cell, 183(7), 1930-1945.e23.

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