Advanced dmem f12

Manufactured by Merck Group

Sourced in Canada, United States, Germany

Advanced DMEM/F12 is a cell culture medium formulated for the growth and maintenance of a variety of cell types. It is a modification of the Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture, providing a balanced set of nutrients and growth factors to support cell proliferation and viability.

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Lab products found in correlation

Advanced dmem f12, by Thermo Fisher Scientific (5 mentions) Glutamax, by Thermo Fisher Scientific (5 mentions) N acetylcysteine, by Merck Group (3 mentions) Fetal bovine serum (fbs), by Merck Group (3 mentions) Glutamax 1, by Merck Group (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Noggin, by R&D Systems (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Matrigel, by BD (2 mentions) Hepes, by Merck Group (2 mentions) Streptomycin, by Merck Group (2 mentions) L glutamine, by Merck Group (2 mentions) Dbcamp, by Merck Group (2 mentions) Matrigel, by Corning (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Cell culture insert, by Merck Group (1 mentions) Red blood cell lysis buffer, by Merck Group (1 mentions) 70 μm cell strainer, by BD (1 mentions) Flagellin, by InvivoGen (1 mentions) Adenosine, by Merck Group (1 mentions)

15 protocols using advanced dmem f12

Retinal Explant Preparation and Culture

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Retinal explants were prepared from 4-wk-old rho^−/− and rho^−/−sarm1^−/− mice and 6 wk-old C57BL/6J and sarm1^−/− mice. Eyes were enucleated and the cornea and lens were carefully removed. Retinas were carefully separated from the RPE/choroid, and four small incisions were made to flatten out the retina. The retinas were placed photoreceptor-side down on a cell culture insert (Millipore) placed inside a 35-mm dish with 1.2 ml of advanced DMEM-F12 (Sigma-Aldrich) supplemented with 1% penicillin/streptomycin. Retinal explants from rho^−/− and rho^−/−sarm1^−/− mice were cultured for 4 h, and explants from C57BL/6J and sarm1^−/− mice were cultured for 24 h treated with CCCP (50 μM) or DMSO vehicle before FLIM analysis.

Ozaki E., Gibbons L., Neto N.G., Kenna P., Carty M., Humphries M., Humphries P., Campbell M., Monaghan M., Bowie A, & Doyle S.L. (2020). SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration. Life Science Alliance, 3(5), e201900618.

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Murine PDX Model of Tumor Growth

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All murine studies were conducted under an MSK IACUC approved protocol. Patient-derived xenograft models were generated as previously described^{9 (link)}. Briefly, tumor tissues were minced to small pieces and digested in dissociation buffer containing 225U/ml Collagenase type III at 37°C for 3 hours. Samples were then washed with advanced DMEM/F12 (Life Technologies), treated with Red Blood Cell Lysis Buffer (Sigma) for 2 minutes, washed again, filtered through 70μm cell strainers, and cultured in F10 medium (advanced DMEM/F12 with 10% FBS, 1X L-glutamine, 1X sodium pyruvate, 1X non-essential amino acids and 1X penicilin/streptomycin). 2 million cells resuspended in PBS: matrigel (1:1) were injected into the flank of the mice subcutaneously. 2 flank tumors were placed per mouse. In vivo treatment was started when tumor volumes reached 100–150 mm³. Mice were then randomly assigned into vehicle and treatment groups (5 mice, 9–10 tumors in each arm) and were treated with either rapamycin (7.5 mg/kg) or vehicle (0.25% PEG-400, 0.25% Tween-80) every other day by intraperitoneal injection as previously reported^{10 (link)}. Following guidelines set forth by the IACUC, mice were sacrificed when the tumor length reached 2 cm. Tumor volume was measured using calipers (calculated as 0.5 × L × W²), and growth curves were generated using GraphPad Prism software.

Dong Y., Eskandari R., Ray C., Granlund K.L., Santos-Cunha L.D., Miloushev V.Z., Tee S.S., Jeong S., Aras O., Chen Y.B., Cheng E.H., Hsieh J.J, & Keshari K.R. (2018). Hyperpolarized MRI visualizes Warburg effects and predicts treatment response to mTOR inhibitors in patient-derived ccRCC xenograft models. Cancer research, 79(1), 242-250.

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Intestinal Crypt Isolation and Organoid Culture

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Mouse intestinal crypts isolation and organoid culture were performed as previously described^{36 (link)}. Briefly, mouse intestine was cut longitudinally and washed three times with cold PBS. Villi were carefully scraped away and small pieces (5 mm) of intestine were incubated in 0.5 M EDTA in PBS for 40 min on ice. These pieces were then vigorously suspended in cold PBS and the mixture was passed through 70 μm cell strainer (BD Biosciences). The crypt fraction was enriched through centrifugation (3 min at 300–400 g). Then the crypts were embedded in Matrigel (BD Biosciences) and seeded on 48-well or 24-well plates. The intestinal organoid culture medium consists of Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX-I, N2, B27 and N-acetylcysteine (Sigma-Aldrich) and combination of growth factors including EGF (50 ng/mL, Invitrogen), Noggin (100 ng/mL, R&D) and R-spondin1 (500 ng/mL). The organoids were treated with proper dilution of peptides in the medium.

Liao H., Li X., Zhao L., Wang Y., Wang X., Wu Y., Zhou X., Fu W., Liu L., Hu H.G, & Chen Y.G. (2020). A PROTAC peptide induces durable β-catenin degradation and suppresses Wnt-dependent intestinal cancer. Cell Discovery, 6, 35.

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Elucidating Purinergic Signaling Mechanisms

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Suramin and Reactive-Blue-2 (RB-2) were obtained from MP Biomedical (Santa Ana, CA, United States). DMEM/F12, advanced DMEM/F12, HEPES, L-glutamine, penicillin, streptomycin, FBS, Dulbecco’s PBS, apyrase, formyl-methionine-leucyl-phenylalanine (FMLP), LPSs, adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), uridine 5’-triphosphate (UTP), uridine 5’-diphosphate (UDP), adenosine, Zm 241385, and fibroblast growth factor 2 (FGF2) were purchased from Sigma–Aldrich (Oakville, ON, Canada). Collagenase type I, SuperScript III, gentamicin, B-27 and N-2 supplements, polyinosinic–polycytidylic acid [poly(I:C)], EDTA, and TRIzol were obtained from Invitrogen (Carlsbad, CA, United States). Collagen type I was purchased from BD Bioscience (San Jose, CA, United States). Y-27632, mrEGF, Wnt-3a, and R-spondin were purchased from R&D Systems (Minneapolis, MN, United States). Noggin and M-CSF were purchased from PeproTech (QC, Canada). SYBR Green and DNAseI were from Roche Diagnostics (Indianapolis, IN, United States). Flagellin was obtained from InvivoGen (San Diego, CA, United States). Oligo(dt)18 was obtained from Fisher Scientific (Ottawa, ON, Canada). MRS 2500, MRS 2179, MRS 2578, AR-C 118925XX, PSB 1114, and 5-BDBD were purchased from Tocris Bioscience (Minneapolis, MN, United States).

Salem M., Tremblay A., Pelletier J., Robaye B, & Sévigny J. (2018). P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells. Frontiers in Pharmacology, 9, 149.

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Neuronal Induction of iPSCs

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Neuronal induction of iPSCs was performed using a previously described method (Sato et al., 2021 (link)), with slight modifications. Briefly, iPSCs were seeded on iMatrix511-coated 12-well plates at a density of 1.0–1.5 × 10⁵ cells/well in StemFit AK02 N medium. After 3 d, neural induction was initiated by changing the medium to neural induction medium [consisting of Advanced DMEM/F-12 (Thermo Fisher Scientific), 2% B27 supplement (–vitamin A; Thermo Fisher Scientific)] with 150 nm LDN193189 (StemRD), 5 μm SB431542 (Tocris), and 3 μm IWR1e (Calbiochem). On day 6, the cells were dissociated into single cells using Accutase (Nacalai) and seeded onto poly-L-ornithine-coated and laminin-coated 12-well plates at a 1:1–1:2 ratio. On day 12, the cells were dissociated again, and 8 × 105 cells seeded in each well of poly-L-ornithine-coated and laminin-coated six-well plates and cultured in neuronal medium [Advanced DMEM/F-12, 2% B27 supplement, 200 μm ascorbic acid (Sigma), and 200 μm dbcAMP (Sigma)] with 20 μm DAPT (Sigma). On day 18, DAPT was removed, and 10 ng/ml BDNF (Alomone Labs), 10 ng/ml GDNF (Alomone Labs), and 1 μm PD0332991 (Sigma) were added. The medium was changed every 3 d.

Imaizumi K., Ideno H., Sato T., Morimoto S, & Okano H. (2022). Pathogenic Mutation of TDP-43 Impairs RNA Processing in a Cell Type-Specific Manner: Implications for the Pathogenesis of ALS/FTLD. eNeuro, 9(3), ENEURO.0061-22.2022.

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Dissociation and Culturing of Intestinal Organoids

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To dissociate the crypts, the small intestine was incubated at 4 °C in EDTA (5 mM) for 30 min, and for mouse intestinal organoids 100 crypts per well were suspended in Matrigel composed of 50% advanced DMEM/ F12 medium (Gibco) and 50% growth-factor-reduced Matrigel (Corning). After the Matrigel polymerized, complete ENR medium containing advanced DMEM/ F12 (Sigma), 2 mM Glutamax (Invitrogen), 10 mM HEPES (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma), 1 mM N-acetyl cysteine (Sigma), N2 supplement (Invitrogen), 50 ng ml−1 mouse EGF (Peprotech), 100 ng/ml mouse Noggin (Peprotech) and 10% human R-spondin-1-conditioned medium from R-spondin-1-transfected HEK 293T cells were added to the cultures. The media was replaced every 2–3 days. Along with medium changes, treatment wells received WT IL-22 (10 nM) or 22-B3 (10 nM).

Saxton R.A., Henneberg L.T., Calafiore M., Su L., Jude K.M., Hanash A.M, & Garcia K.C. (2021). The Tissue Protective Functions of Interleukin-22 can be Decoupled from Pro-Inflammatory Actions Through Structure-Based Design. Immunity, 54(4), 660-672.e9.

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Isolation and Culture of Mouse Ear Fibroblasts

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MCF-7 cells (ATCC), were cultivated as described in [3 (link)]. Primary mouse ear fibroblast were isolated and cultivated as follows:
Mouse ear notches or whole ears were collected and stored in cold Serum Free Medium (SFM), Advanced DMEM/F12 (Life Technologies) chopped with a sterile scalpel and incubated in SFM with 1mg/mL of collagenase A (Roche) for 1.5h at 37°C in 5% O₂. After digestion, the tissue was passed through an 18G syringe needle and then centrifuged for 10 minutes at 1000 rpm. Next, pellets were resuspended in Advanced DMEM/F12 medium with 10% FBS (SIGMA), 2mM L-glutamine (GIBCO) and a mix of 50U/mL penicillin and 10mg/mL streptomycin (GIBCO). Isolated cells were incubated and cultivated at 37°C in 3% O₂ and 5% CO₂.
Cells were treated with 100 or 200nM rapamycin (SIGMA) in serum containing medium for 3 days. Controls were treated with the same amount of DMSO. Bosutinib (Santa Cruz Biotechnology) was also prepared in DMSO and controls treated with DMSO only.

Miwa S., Czapiewski R., Wan T., Bell A., Hill K.N., von Zglinicki T, & Saretzki G. (2016). Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria. Aging (Albany NY), 8(10), 2551-2564.

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Establishing Patient-Derived CRC Organoids

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CRC patient tumor tissues were washed with cold HBSS. After removing the muscle tissue, the epithelial tumor tissues were cut into small pieces and vigorously suspended. Then the tissue suspension was centrifuged (3 min at 300–400 g) and tumor fraction was enriched at the bottom. Next, the tumor fraction was embedded in Matrigel (BD Biosciences) and seeded on 24-well plates with the culture medium, which includes Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine (Sigma-Aldrich), CHIR-99021 (5 μM, Selleck), A-83–01 (0.5 μM, Cayman), SB202190 (10 μM, Selleck), gastrin (1 nM, Tocris), Y27632 (10 μM, Enzo), PEG2 (2.5 μM, Selleck), nicotinamide (10 mM, Sigma-Aldrich), EGF (50 ng/mL, Invitrogen), Noggin (100 ng/mL, R&D) and R-spondin1 (500 ng/mL).

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Colon Crypt Isolation and 3D Organoid Culture

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Crypts were separately isolated from proximal and distal halves of the colon as described previously [39 (link)]. Briefly, crypts were isolated by digesting the tissue with 500 U/ml collagenase XI (Sigma), 0.4 U/ml dispase (Roche), and 1 mM dithiothreitol. Obtained crypts were suspended in the collagen type I solution (Nitta Gelatin Inc.) and placed in 24-well plates. After polymerization, 500 μl of Advanced DMEM/F12 containing 1% BSA (Sigma), 30 ng/ml mWnt3a, 500 ng/ml mRspo1, 50 ng/ml mHGF, 50 ng/ml mNoggin (all from R&D Systems), and 20 ng/ml mEGF (Peprotech) were added to each well. When indicated, organoids were treated with either 1 μM LY411575 (Santa Cruz Biotechnology), a GSI, or vehicle (DMSO) alone for 48 hours from day 3 to day 5 of culture.

Akiyama S., Mochizuki W., Nibe Y., Matsumoto Y., Sakamoto K., Oshima S., Watanabe M, & Nakamura T. (2017). CCN3 Expression Marks a Sulfomucin-nonproducing Unique Subset of Colonic Goblet Cells in Mice. Acta Histochemica et Cytochemica, 50(6), 159-168.

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Intestinal Tumor Spheroid Culture

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The extracted mice intestines were washed several times with PBS and the intestinal tumors were dissected. Tumor cells were dissociated using 2.5 mg collagenase from Clostridium histolyticum (Sigma‐Aldrich) with 2.5 ml advanced DMEM/F‐12 (Thermo Fisher Scientific). After dissociation, tumor cells were collected by centrifugation and embedded in Matrigel (Corning). For spheroid culture of tumor cells, advanced DMEM/F‐12 and 10% FBS (Sigma‐Aldrich) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (FUJIFILM Wako Pure Chemical Corporation), and GlutaMAX (Thermo Fisher Scientific) was added to each well. To establish the spheroids (P0), 10 mmol/L Y‐27632 (Tocris Bioscience) was added to the culture medium. For spheroid culture of intestinal tumors of Dclk1^{CreERT2‐IRES‐EGFP/+}; Apc^Min/+; Brg1^flox/flox spheroid, 20 ng/ml murine interleukin‐13 (PeproTech) and 1 mM valproic acid (Fujifilm Wako Pure Chemical Corporation) was added to the culture medium.
For induction of Cre‐mediated recombination in vitro, 1 μmol/L 4‐OHT (Sigma‐Aldrich) was added to the culture medium.

Yoshikawa T., Fukuda A., Omatsu M., Namikawa M., Sono M., Fukunaga Y., Masuda T., Araki O., Nagao M., Ogawa S., Masuo K., Goto N., Hiramatsu Y., Muta Y., Tsuda M., Maruno T., Nakanishi Y., Kawada K., Takaishi S, & Seno H. (2022). JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression. Cancer Science, 113(10), 3417-3427.

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