mem non essential amino acid, by Corning - Product details

1

Mycobacterium tuberculosis Culture and Ex Vivo Macrophage Assay

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Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).
Ex vivo MΦ were enriched from mice via bronchoalveolar lavage or peritoneal lavage with DMEM + 10% FBS + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin + 100 mg/mL Streptomycin (Sigma P4333). Lavage cells were treated with ACK lysis buffer (0.15M NH₄Cl, 10mM KHCO₃, 0.1mM EDTA) to lyse red blood cells, plated in tissue culture treated plates, and incubated at 37°C in 5% CO₂ for at least 4 hours to allow adherence of MΦ^{14 (link)}. Wells were washed vigorously with PBS to remove non-adherent cells and lysed in 2X Laemmli buffer for western blot analysis.
Bone marrow derived MΦ were isolated from femurs and tibias of mice, and cultured in DMEM + 20% FBS + 10% supernatant from 3T3 cells overexpressing M-CSF + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin and 100 μg/mL Streptomycin (Sigma P4333) at 37° C in 5% CO₂.
PMN for ex vivo western blotting analysis were purified from uninfected bone marrow by negative selection via MACS column (Miltenyi Biotech, 130-097-658) according to manufacturer’s guidelines and immediately lysed in 2X Laemmi buffer.

Kimmey J.M., Huynh J.P., Weiss L.A., Park S., Kambal A., Debnath J., Virgin H.W, & Stallings C.L. (2015). Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection. Nature, 528(7583), 565-569.

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Mycobacterium tuberculosis Culture and Ex Vivo Macrophage Assay

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Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).
Ex vivo MΦ were enriched from mice via bronchoalveolar lavage or peritoneal lavage with DMEM + 10% FBS + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin + 100 mg/mL Streptomycin (Sigma P4333). Lavage cells were treated with ACK lysis buffer (0.15M NH₄Cl, 10mM KHCO₃, 0.1mM EDTA) to lyse red blood cells, plated in tissue culture treated plates, and incubated at 37°C in 5% CO₂ for at least 4 hours to allow adherence of MΦ^{14 (link)}. Wells were washed vigorously with PBS to remove non-adherent cells and lysed in 2X Laemmli buffer for western blot analysis.
Bone marrow derived MΦ were isolated from femurs and tibias of mice, and cultured in DMEM + 20% FBS + 10% supernatant from 3T3 cells overexpressing M-CSF + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin and 100 μg/mL Streptomycin (Sigma P4333) at 37° C in 5% CO₂.
PMN for ex vivo western blotting analysis were purified from uninfected bone marrow by negative selection via MACS column (Miltenyi Biotech, 130-097-658) according to manufacturer’s guidelines and immediately lysed in 2X Laemmi buffer.

Kimmey J.M., Huynh J.P., Weiss L.A., Park S., Kambal A., Debnath J., Virgin H.W, & Stallings C.L. (2015). Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection. Nature, 528(7583), 565-569.

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3

Cell Culture and ChIP Assay Protocol

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MCF7 (ATCC, HTB-22) and HEPG2 (ATCC, HB-8065) were grown in DMEM (Gibco, 11965–092). A549 (ATCC, CCL-185) were grown in F12K medium (Gibco, 21127022). Culture medium were supplemented with 10% fetal bovine serum (Invitrogen, qualified 12483020), 100 μM MEM nonessential amino acids (Cellgro, 25–0250), 2 mM L-glutamine (Gibco, 25030–081), 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco, 15170–063).
For ChIP experiments, MCF7 were kept the last 3 days into DMEM w/o phenol-red (Gibco, 31053–028) supplemented with 5% of Charcoal/Dextran treated FBS (Hyclone, AVH78911), 100 μM MEM nonessential amino acids (Cellgro, 25–0250), 2 mM L-glutamine (Gibco, 25030–081) and treated with 100 nM of Beta-Estradiol (SIGMA, E8875) for two hours.

Fournier M., Bourriquen G., Lamaze F.C., Côté M.C., Fournier É., Joly-Beauparlant C., Caron V., Gobeil S., Droit A, & Bilodeau S. (2016). FOXA and master transcription factors recruit Mediator and Cohesin to the core transcriptional regulatory circuitry of cancer cells. Scientific Reports, 6, 34962.

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Culture and Maintenance of Human Cell Lines

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Human pre-B cell lines Nalm 6 and N114P2 (Lig IV−/−) were obtained from Dr. M.R. Lieber (University of Southern California, Los Angeles, CA (22 (link)). Growth medium and conditions for pre-B cells were as previously described (23 (link)). LIG4 (GM16088 and GM17523B) (24 (link)) and Ligase I (GM16096 and GM16097A) (25 (link)) human hypomorphic fibroblast cells were obtained from Coriell Cell Repositories. NM-1, wild type human fibroblasts were obtained from Dr. A. Villa (Unita di Milano, Milan, Italy) and HFF-1, wild type human foreskin fibroblasts were obtained from the American Type Culture Collection. Cells were maintained at 37°C and 5% CO₂. Wild type human fibroblasts were cultured on plates pretreated with 0.2% gelatin in DMEM, 10% heat inactivated Fetal Bovine Serum (Invitrogen), 1% antibiotic-antimycotic, 1 mM sodium pyruvate, 2 mM glutamate, 1% MEM non-essential amino acids (Cellgro), 20 mM HEPES (Cellgro), and 100 µM β-mercaptoethanol (β-ME) (Sigma). LIG4 Syndrome and Ligase I hypomorphic fibroblasts were grown in Minimum Essential Medium (Sigma), 15% Fetal Bovine Serum (Invitrogen), 1% antibiotic-antimycotic, 1 mM sodium pyruvate, 2 mM glutamate, and 1% MEM non-essential amino acids (Cellgro).

Francis D.B., Kozlov M., Chavez J., Chu J., Malu S., Hanna M, & Cortes P. (2014). DNA Ligase IV regulates XRCC4 nuclear localization. DNA repair, 21, 36-42.

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5

Cell Culture Protocols for Immune Cells

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Vero and A549 cells were obtained from ATCC and maintained in complete DMEM (DMEM medium [Corning] supplemented with 10% fetal bovine serum [Optima, Atlanta Biologics], 2mM L-Glutamine [Corning], 1mM HEPES [Corning], 1mM sodium pyruvate [Corning], 1x MEM Non-essential Amino Acids [Corning], and 1x Antibiotics/Antimycotics [Corning]). moDCs, monocytes, mDCs and pDCs were maintained in complete RPMI (RPMI 1640 medium [Corning] supplemented with 10% fetal bovine serum [Optima, Atlanta Biologics], 2mM L-Glutamine [Corning], 1mM sodium pyruvate [Corning], 1x MEM Non-essential Amino Acids [Corning], and 1x Antibiotics/Antimycotics [Corning]).

Bowen J.R., Quicke K.M., Maddur M.S., O’Neal J.T., McDonald C.E., Fedorova N.B., Puri V., Shabman R.S., Pulendran B, & Suthar M.S. (2017). Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells. PLoS Pathogens, 13(2), e1006164.

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6

Plasma Cell and IgG Differentiation of Naive B Cells

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CD43-depleted naive B cells were labeled with eFluor 670 (Thermo Fisher Scientific) according to the manufacturer’s protocol and seeded at a final concentration of 0.2 × 10⁶ cells/ml. For plasma cell differentiation, the B cells were stimulated with 5 μg/ml LPS (Sigma-Aldrich) for 72 h. For IgG1 or IgG3 CSR, the B cells were stimulated with 5 μg/ml anti-CD40 agonistic antibody (clone: 1C10; Thermo Fisher Scientific) and 2.5 ng/ml recombinant mouse IL-4 (Thermo Fisher Scientific) or 5 μg/ml LPS (Sigma-Aldrich) for 96 h, respectively. All B cells were cultured in complete Roswell Park Memorial Institute 1640 (Cellgro; Corning) supplemented with 10% FBS (Sigma-Aldrich), 1× penicillin/streptomycin (Cellgro; Corning), 2 mM GlutaGro (Cellgro; Corning), 1× MEM nonessential amino acids (Cellgro; Corning), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 50 mM β-mercaptoethanol (Thermo Fisher Scientific).

Zhu Z., Shukla A., Ramezani-Rad P., Apgar J.R, & Rickert R.C. (2019). The AKT isoforms 1 and 2 drive B cell fate decisions during the germinal center response. Life Science Alliance, 2(6), e201900506.

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7

Dendritic Cell Generation from Murine Bone Marrow

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DCs were generated from murine bone marrow cells, as described by Garrigan et al^{23 (link)} with minor modifications. Briefly, bone marrow was flushed from the long bones of C57BL/6 mice. A single cell suspension was cultured in RPMI 1640 (CellGro, USA) supplemented with 10% heat-inactivated FBS (Life Technologies, USA), 2 mM L-glutamine (Gibco, USA), 100 U/mL penicillin (Gibco, USA), 100 µg/mL streptomycin (Gibco, USA), 10 mM HEPES pH 7.4 (Gibco, USA), 0.5 mM sodium pyruvate (Cellgro, USA), 0.5% MEM non-essential amino acids (Cellgro, USA), 0.1 mg/mL Normocin (InVivogen, USA), along with 20 ng/mL GM-CSF (Peprotech) and 10 ng/mL IL-4 (Peprotech, USA) for 4-DCs or 20 ng/mL GM-CSF and 250 U/mL IFNα (AbD Serotec, USA) for α-DCs. On day 2, the medium was replaced with fresh medium containing the respective cytokines. On day 4, 100 µL of the indicated tumor lysate preparations were added to 10 mL culture (~1 tumor cell equivalent per DC) and incubated overnight at 37°C and 5% CO₂. Unpulsed DCs were also cultured as negative controls. On day 5, the medium was removed and fresh medium containing 100 ng/mL LPS (Sigma-Aldrich, Germany) and 100 ng/mL IFNγ (Peprotech, USA) was added. DCs were then cultured overnight at 37°C and 5% CO₂.

Mookerjee A., Graciotti M, & Kandalaft L.E. (2018). A cancer vaccine with dendritic cells differentiated with GM-CSF and IFNα and pulsed with a squaric acid treated cell lysate improves T cell priming and tumor growth control in a mouse model. BioImpacts : BI, 8(3), 211-221.

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8

Culturing Human Uveal Melanoma Cell Lines

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All cell lines were cultured at 37 °C, 5% CO₂ in a humidified incubator according to the standard protocol of mammalian cell culturing. Human UM cells 92.1, OCM1, OMM2.3, OMM3, M20-07-070, M20-09-196, Mel290, and Mel270, authenticated by STR (Emory Genomics core facility) [22 (link)] were cultured in RPMI 1640 with L-glutamine (Corning Cellgro, Albany, NY) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), Sodium Pyruvate (Cellgro, Albany, NY), MEM Non-Essential Amino Acids (Cellgro, Albany, NY), MEM Vitamins (Cellgro, Albany, NY), Penicillin-Streptomycin Solution (Cellgro, Albany, NY), and HEPES buffer (Corning, Albany, NY). The 92.1 and OMM2.3 cells were provided by Dr. Jerry Niederkorn (Department of Ophthalmology, UT Southwestern, Dallas, TX). The Mel290 and Mel270 cells were provided by Dr. Bruce Ksander (Schepens Eye Institute, Boston, MA). The OCM1 and OMM3 cells were donated by Dr. June Kan-Mitchel (Wayne State University, Detroit, MI). Dr. Scott Woodman (Department of Melanoma Medical Oncology and Systems Biology, MD Anderson Cancer Center, Houston, TX), Dr. Barry Burgess, and Dr. Tara McCannel (UCLA, Jules Stein Eye Institute, Calabasas, CA) isolated and provided M20-09-196 and M20-07-070 cell lines. SKOV3 ovarian cancer cells were cultured in McCoy 5 A medium with 10% fetal bovine serum and 1% penicillin and streptomycin.

Yang H., Tan S., Qiao J., Xu Y., Gui Z., Meng Y., Dong B., Peng G., Ibhagui O.Y., Qian W., Lu J., Li Z., Wang G., Lai J., Yang L., Grossniklaus H.E, & Yang J.J. (2022). Non-invasive detection and complementary diagnosis of liver metastases via chemokine receptor 4 imaging. Cancer Gene Therapy, 29(12), 1827-1839.

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9

Foreskin Fibroblast Cell Culture

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Low-passage primary human foreskin fibroblasts (HFF) BJ strain (ATCC CRL-2522) were propagated in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gemini Bioproducts), 1x MEM Non-essential Amino Acids (Cellgro) and 100 units Penicillin/100 μg Streptomycin/0.25 μg Amphotericin per mL (DMEM-10% FBS) and incubated at 37°C in 5% CO₂. Following expansion, 2.5x10⁵ cells were seeded into 6-well plates in DMEM-10% FBS for 48 hours. Identical wells were prepared for microscopy with glass microscope coverslips for parasite burden computation. Two biological replicates, each originating from separate freeze dates after expansion from the original ATCC culture, were performed.

Houston-Ludlam G.A., Belew A.T, & El-Sayed N.M. (2016). Comparative Transcriptome Profiling of Human Foreskin Fibroblasts Infected with the Sylvio and Y Strains of Trypanosoma cruzi. PLoS ONE, 11(8), e0159197.

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Culturing Embryonic Stem Cells

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ESCs were cultured on gelatin-coated plates in standard ESC medium consisting of DMEM (Cellgro) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone), 1% Glutamax (GIBCO), 1% Penicillin/Streptomycin (Cellgro), 1% nucleoside (Millipore), 0.1mM 2-mercaptoethanol (GIBCO), 1% MEM nonessential amino acids (Cellgro), and 1000U/ml recombinant leukemia inhibitory factor (Millipore).

Lu J.Y., Shao W., Chang L., Yin Y., Li T., Zhang H., Hong Y., Percharde M., Guo L., Wu Z., Liu L., Liu W., Yan P., Ramalho-Santos M., Sun Y, & Shen X. (2020). Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation. Cell reports, 30(10), 3296-3311.e5.

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Mem non essential amino acid

Lab products found in correlation

86 protocols using mem non essential amino acid

Mycobacterium tuberculosis Culture and Ex Vivo Macrophage Assay

Mycobacterium tuberculosis Culture and Ex Vivo Macrophage Assay

Cell Culture and ChIP Assay Protocol

Culture and Maintenance of Human Cell Lines

Cell Culture Protocols for Immune Cells

Plasma Cell and IgG Differentiation of Naive B Cells

Dendritic Cell Generation from Murine Bone Marrow

Culturing Human Uveal Melanoma Cell Lines

Foreskin Fibroblast Cell Culture

Culturing Embryonic Stem Cells

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