NPCs were derived from embryonic stem cell line BG02, provided by Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine [41 (link)]. NPCs culture medium contained Neurobasal medium (Gibco, Thermo Fisher Scientific), 1% N2 (Gibco), 2% B27 (Gibco), 1% NEAA (Sigma), 1% Glutmax (Sigma), 10 ng/mL bFGF (1:500; Millipore Ltd., Burlington, MA, USA), and 1000 U/mL LIF (Millipore), 3 μM CHIR99021 (Selleck, Houston, TX, USA), and 5 μM SB431542 (Cellagentec, San Diego, CA, USA). NPCs were cultured with 5 μg/mL laminin (Gibco) in poly-ornithine-coated 12-well plates and were passaged for every three days during the experiment [41 (link)]. Staining was carried out with PAX6 (1:500, Biolegend Covance, Dedham, MA, USA), SOX2 (1:500, Millipore), and Nestin (1:200, Millipore) [42 (link)], which are specific NSC markers. Primary cortical neuron culture was prepared from cerebral cortical tissue from postnatal day one (P1) wild-type C57BL/6 mice. The tissue was digested with 0.25% trypsin for 30 min followed by centrifugation at 1000 rpm for 5 min and filtering cells with 40 μm filter. Astrocytes culture medium contained DMEM (Gibco), 10% fetal bovine serum (FBS, Northvale, NJ, USA), and 1% NEAA (Sigma). Neuron culture medium contained Neurobasal (Gibco), 2% B27 (Gibco), 1% N2 (Gibco), and 1% NEAA (Sigma).
Non essential amino acids (neaa)
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Non-essential amino acids are a class of amino acids that can be synthesized by the human body and are not required to be obtained from the diet. They play a crucial role in various biological processes and are an important component of proteins.
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Lab products found in correlation
L glutamine, by Merck Group (217 mentions)
Sodium pyruvate, by Merck Group (166 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (152 mentions)
Penicillin streptomycin, by Merck Group (139 mentions)
Penicillin, by Merck Group (131 mentions)
Streptomycin, by Merck Group (129 mentions)
Fetal bovine serum (fbs), by Merck Group (129 mentions)
β mercaptoethanol, by Merck Group (80 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (79 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (79 mentions)
L glutamine, by Thermo Fisher Scientific (73 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (73 mentions)
Penicillin, by Thermo Fisher Scientific (72 mentions)
Glutamax, by Thermo Fisher Scientific (70 mentions)
Streptomycin, by Thermo Fisher Scientific (66 mentions)
Hepes, by Merck Group (55 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (38 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (37 mentions)
2 mercaptoethanol, by Merck Group (36 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (36 mentions)
905 protocols using non essential amino acids (neaa)
1
Neural Progenitor Cell Derivation and Characterization
2
Murine Cell Culture Protocol
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The murine endothelial cell line bEnd5 was obtained from the HPA Culture Collections via Sigma-Aldrich in August 2018. Experiments with these cells were completed within 3 months and the cells were therefore not authenticated again. bEnd5 cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, USA) containing 1% sodium pyruvate (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich). Fibroblast 3T3 cells were cultured in DMEM/F-12 medium (Thermo Fisher Scientific). Murine breast cancer cells (PyMT) were cultured in DMEM containing 1% sodium pyruvate, 1% non-essential amino acids, and 10 mmol/L HEPES (Sigma-Aldrich). Media was supplemented with 10% FCS (Capricorn Scientific, Epsdorfergrund, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin (PAA laboratories, Cölbe, Germany).
3
Culturing HEK293T, MEFs, and iPSCs
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HEK293T cells, mouse embryonic fibroblasts (MEFs), and OG2 MEFs were cultured in low‐glucose (1,000 mg/l) Dulbecco's modified Eagle's medium (DMEM, Sigma‐Aldrich), supplemented with 10% fetal bovine serum (FBS, Biochrom), 2 mM l ‐glutamine and 1× penicillin/streptomycin (Sigma‐Aldrich), 1% non‐essential amino acids (Sigma‐Aldrich), and 0.10 mM β‐mercaptoethanol. OG2‐MEFs were isolated as described previously 1 . Mouse embryonic stem cells (mESCs) and iPSCs were maintained in high‐glucose (4,500 mg/l) DMEM (Sigma‐Aldrich), supplemented with 10% fetal bovine serum (Biochrom), 5% serum replacement (Gibco), 2 mM l ‐glutamine, 1× penicillin/streptomycin (Sigma‐Aldrich), 1% non‐essential amino acids (Sigma‐Aldrich), 1% sodium pyruvate (Sigma‐Aldrich), 0.10 mM β‐mercaptoethanol (Gibco) with 1,000 units of leukemia inhibitory factor (LIF; prepared in house) on a feeder layer of gamma‐irradiated MEFs; experiments under feeder‐free conditions were performed using mESC medium and 2,000 units of LIF, as previously described 67 .
4
Cell Culture Protocols for Diverse Cell Lines
5
Cell Culture and Transfection Protocols
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NIH3T3 mouse fibroblasts and HEK293T human epithelial cells were obtained from the American Type Culture Collection (ATCC). The vinculin null and WT mouse embryonic fibroblasts (MEFs) originate from the Eileen Adamson laboratory [32 (link)]. All cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma) and 1% non-essential amino acids (Sigma). Talin1&2 null cells [12 (link)] were cultured in DMEM F-12 (Gibco) supplemented with 10% FBS, 1% L-glutamine, 1% non-essential amino acids and 15 µM HEPES (Sigma). All cells were cultured at 37̊C supplied with 5% CO2 and 95% humidity. Transient transfections were performed using Lipofectamine LTX with Plus Reagent (Invitrogen) to NIH3T3 cells, Lipofectamine 2000 (Invitrogen) to talin null cells, and jetPRIME reagent (Polyplus) to MEFs and HEK293T cells, as per the manufacturer's instructions.
6
Cell Culture Protocols for Diverse Cell Lines
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CHO (Chinese hamster ovary) cells were maintained in Alpha-MEM medium
(Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS, Gibco) and 2 mM
L-glutamine (Sigma-Aldrich). HEK293 (human embryonic kidney, ATCC) cells were
grown in DMEM (Dulbecco’s modified Eagle’s medium, Sigma-Aldrich)
supplemented with 10% FBS and 2 mM L-glutamine. MDA-MB-231 (triple-negative
human breast adenocarcinoma, ATCC) cells were maintained in DMEM supplemented
with 10% FBS, 2 mM L-glutamine, and 1% non-essential amino acids
(Sigma-Aldrich). Immortalized mouse embryonic fibroblasts (MEFs) isolated from
wild-type and SHARPIN-null (cpdm) mice have been described
previously 7 (link). MEFs were cultured in DMEM
supplemented with 10% FBS, 2 mM L-glutamate, 1 mM sodium pyruvate
(Sigma-Aldrich), 1% non-essential amino acids and 1:100000
β-mercaptoethanol (Sigma-Aldrich). SK-N-BE-2 neuroblastoma cells (ATCC)
were maintained in DMEM:HAM’s F-12 medium supplemented with 10% FBS, 2 mM
glutamine and 1% non-essential amino acids. All cells were routinely tested for
mycoplasma contamination. No cell lines used in this study were found in the
database of commonly misindentified cell lines that is maintained by ICLAC and
NCBI Biosample. The cell lines were not authenticated.
(Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS, Gibco) and 2 mM
L-glutamine (Sigma-Aldrich). HEK293 (human embryonic kidney, ATCC) cells were
grown in DMEM (Dulbecco’s modified Eagle’s medium, Sigma-Aldrich)
supplemented with 10% FBS and 2 mM L-glutamine. MDA-MB-231 (triple-negative
human breast adenocarcinoma, ATCC) cells were maintained in DMEM supplemented
with 10% FBS, 2 mM L-glutamine, and 1% non-essential amino acids
(Sigma-Aldrich). Immortalized mouse embryonic fibroblasts (MEFs) isolated from
wild-type and SHARPIN-null (cpdm) mice have been described
previously 7 (link). MEFs were cultured in DMEM
supplemented with 10% FBS, 2 mM L-glutamate, 1 mM sodium pyruvate
(Sigma-Aldrich), 1% non-essential amino acids and 1:100000
β-mercaptoethanol (Sigma-Aldrich). SK-N-BE-2 neuroblastoma cells (ATCC)
were maintained in DMEM:HAM’s F-12 medium supplemented with 10% FBS, 2 mM
glutamine and 1% non-essential amino acids. All cells were routinely tested for
mycoplasma contamination. No cell lines used in this study were found in the
database of commonly misindentified cell lines that is maintained by ICLAC and
NCBI Biosample. The cell lines were not authenticated.
7
Propagation of Zika Virus Variants
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Vero (a kidney epithelial cell line from an African green monkey) and A549 (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the American Type Culture Collection (ATCC, CCL-81) and were grown and maintained at 37 °C and 5% CO2 in growth medium, consisting in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) (HyClone, ThermoFisher Scientific, Madrid, Spain), 2 mM l -glutamine (Sigma-Aldrich, Madrid, Spain), 1% nonessential amino acids (Sigma-Aldrich), 100 U/mL penicillin (Sigma-Aldrich) and 100 µg/mL streptomycin (Sigma-Aldrich).
The recombinant ZIKV-RGN WT (rZIKV-RGN) or NS2A mutant (rZIKV-RGN-mNS2A) viruses were propagated in Vero cells with virus growth medium (DMEM supplemented with 2% FBS, 2 mMl -glutamine, 1% nonessential amino acids, 100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C and 5% CO2. For virus stocks preparation, 80 to 90% confluent monolayers of Vero cells were infected with a multiplicity of infection (MOI) of 0.1 plaque forming units (PFU) per cell in virus growth medium and incubated at 37 °C under 5% CO2. After 3–4 days of infection, the tissue culture supernatants were collected, clarified by centrifugation at 6000× g for 5 min, and stored in small aliquots at −80 °C.
The recombinant ZIKV-RGN WT (rZIKV-RGN) or NS2A mutant (rZIKV-RGN-mNS2A) viruses were propagated in Vero cells with virus growth medium (DMEM supplemented with 2% FBS, 2 mM
8
Cell Culture Protocols for SARS-CoV-2 Research
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Vero E6 cells (Cercopithecus aethiops -derived epithelial kidney cells, ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, catalog no. 41965039) supplemented with 2.5% heat-inactivated fetal calf serum (FCS; Gibco, catalog no. 10270106), 100 units/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher, catalog no. 15140122), 2 mM l -glutamine, 1 mM sodium pyruvate (Pan Biotech, catalog no. P04-8010), and 1× nonessential amino acids (Sigma, catalog no. M7145). Caco-2 cells (human epithelial colorectal adenocarcinoma cells, kindly provided by H. Barth, Ulm University) were grown in the same media but with supplementation of 10% FCS. Calu-3 cells (human epithelial lung adenocarcinoma cells, kindly provided by M. Frick, Ulm University) were cultured in minimum essential Eagle medium (MEM; Sigma, catalog no. M4655) supplemented with 10% FCS (during viral infection) or 20% FCS (at all other times), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 1× nonessential amino acids. NHLF cells (primary human lung fibroblasts; Lonza), HEK293T ZAP KO cells (10 (link)), and A549 cells (adenocarcinoma human alveolar basal epithelial cells; ATCC) were cultured in DMEM supplemented with 10% FCS, 2 mM μg/ml l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.
9
Culturing Diverse Cell Lines for Experiments
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Vero E6 (Cercopithecus aethiops derived epithelial kidney) cells were grown in Dulbecc’s modified Eagle’s medium (DMEM) supplemented with 2.5% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and non-essential amino acids (Sigma #M7145, St. Louis, MI, USA). HEK293T cells are a human cell line originally derived from embryonic kidney. Transformed by adenovirus type 5 expressing SV40 (simian virus 40) large T-antigen [11 (link)]. ELVIS™ (Enzyme-Linked Virus-Inducible System) are a baby hamster kidney cell line encoding lacZ gene, which is expressed upon infection via viral transactivator ICP10 [12 (link)].
HEK293T and ELVIS™ cells were cultured in DMEM supplemented with 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin, and 10% heat-inactivated FCS. Caco-2 (human epithelial colorectal adenocarcinoma) cells were grown in DMEM supplemented with 20% FCS, 100 units /mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and non-essential amino acids (Sigma Aldrich, St. Louis, MO, USA) and seeded in the same medium with only 10% FCS for experiments. Cells were grown at 37 °C in a 5% CO2 humidified incubator.
HEK293T and ELVIS™ cells were cultured in DMEM supplemented with 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin, and 10% heat-inactivated FCS. Caco-2 (human epithelial colorectal adenocarcinoma) cells were grown in DMEM supplemented with 20% FCS, 100 units /mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and non-essential amino acids (Sigma Aldrich, St. Louis, MO, USA) and seeded in the same medium with only 10% FCS for experiments. Cells were grown at 37 °C in a 5% CO2 humidified incubator.
10
Comparative Analysis of Cell Lines
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Three cell lines were used, HUVECs, HaCaT and HeLa cells. The HUVEC cell line was cultured in MEM/Earle medium (Cultilab, Brazil), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil), 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA), and 1% l ‐glutamine (200 μm ) (Sigma‐Aldrich, St Louis, MO, USA). The HaCaT cell line was cultured in MEM/Earle medium (Cultilab), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab), 1% antibiotic/antimycotic (Invitrogen), 1% l ‐glutamine (200 μm ) (Sigma‐Aldrich), 1% non‐essential amino acids (10 mm ; Sigma‐Aldrich), and 1% sodium pyruvate (100 mm ; Sigma‐Aldrich). The HeLa cell line were cultured in MEM/Earle medium (Cultilab), pH 7.5, supplemented with 10% fetal bovine serum (Cultilab), 1% antibiotic/antimycotic (Invitrogen) 1% l ‐glutamine (200 μm ; Sigma‐Aldrich), and 1% non‐essential amino acids (10 mm ; Sigma‐Aldrich). A total of 106 cells from each cell line were seeded in 75 cm2 culture flasks and kept at 37 °C in an atmosphere of 5% CO2.
We did all the experiments with another cervix cell line (SiHa – ATCC, Manassas, VA, USA) treated with the peptide, but without the co‐treatment with conditioned medium of endothelial cells (HUVECs), and the results were the same with the HeLa; for this reason we did not continued with the co‐treatment.
We did all the experiments with another cervix cell line (SiHa – ATCC, Manassas, VA, USA) treated with the peptide, but without the co‐treatment with conditioned medium of endothelial cells (HUVECs), and the results were the same with the HeLa; for this reason we did not continued with the co‐treatment.
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