Primary chicken embryonic fibroblasts were prepared for constructing and propagating the recombinant VACV DIs strain that carries the gene encoding the spike protein of SARS-CoV-2. Seven-day-old chicken embryos were collected in Hanks’ Balanced Salt Solution [HBSS (−)] supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, and 0.1% glucose. After removing the eyes, brain, beak, wings, and feet from each embryo, the rest of the body was minced with scissors and digested in TrypLE Select (Thermo Fisher Scientific, Waltham, MA, United States). The resulting chicken embryonic fibroblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal calf serum and tryptose phosphate broth.
Dulbecco s modified eagle medium dmem
Manufactured by Nissui Pharmaceutical
Sourced in Japan
Dulbecco's Modified Eagle Medium (DMEM) is a cell culture medium used to support the growth of a variety of cell types. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell proliferation and maintenance.
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Lab products found in correlation
L glutamine, by Fujifilm (4 mentions)
Streptomycin, by Fujifilm (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Rpmi 1640 medium, by Nissui Pharmaceutical (2 mentions)
Polyethylenimine max, by Polysciences (2 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Fujifilm (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions)
Mcf 7, by RIKEN BioResource Center (1 mentions)
Mda mb 453, by Merck Group (1 mentions)
293ft cell line, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by Nissui Pharmaceutical (1 mentions)
Mda mb 453, by RIKEN BioResource Center (1 mentions)
Mda mb 231, by Merck Group (1 mentions)
Leibovitz l 15 medium, by Merck Group (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Mcf 10a, by Lonza (1 mentions)
14 protocols using dulbecco s modified eagle medium dmem
1
Constructing Recombinant VACV DIs Strain
2
OVA and Fluorescently-Labeled Derivatives Production
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OVA was purchased from Sigma-Aldrich (St. Louis, MO, USA). BK was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). The γ-PGA was provided by Yakult Pharmaceutical Industry Co., Ltd. (Tokyo, Japan). Fluorescein isothiocyanate-labelled OVA (FITC-OVA) and Alexa Fluor 647-labeled OVA (Alexa647-OVA) were obtained from Invitrogen (Carls, CA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel). OPTI-MEM I was obtained from GIBCO BRL (Grand Island, NY, USA), and a premix antibiotics solution containing penicillin, streptomycin, and L-glutamine were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dulbecco’s Modified Eagle Medium (DMEM) and RPMI1640 medium were obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan).
3
Isolation and Culture of Human Oral Fibroblasts
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hOFs were isolated from individually collected buccal mucosal tissues obtained from four healthy volunteers (26–35 years old) after receiving written agreement including an informed consent at the Tokushima University Medical and Dental Hospital. Approval from the Institutional Research Ethics Committee of the University of Tokushima was obtained (Project number 708). Details on hOFs isolation have been described previously [5 (link)]. After isolation, hOFs were individually designated as hOF1 to hOF4. Among them, we failed to establish primary cell culture from hOF1, so we used three successful cell lines, hOF2, hOF3, and hOF4, for further experiments.
hOFs (hOF2, hOF3, and hOF4) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan). Three types of hDFs derived from individuals aged 33–36 years old were purchased from the Health Science Research Resources Bank (TIG110, TIG111, and TIG114; Osaka, Japan). hDFs were cultured in Eagle's MEM (EMEM; Nissui) supplemented with 10% FBS (Nichirei Biosciences).
hOFs (hOF2, hOF3, and hOF4) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan). Three types of hDFs derived from individuals aged 33–36 years old were purchased from the Health Science Research Resources Bank (TIG110, TIG111, and TIG114; Osaka, Japan). hDFs were cultured in Eagle's MEM (EMEM; Nissui) supplemented with 10% FBS (Nichirei Biosciences).
4
Culturing Human Breast Cancer and Mammary Epithelial Cell Lines
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Human breast cancer cell lines, MCF-7 and MDA-MB-453, were provided by the RIKEN BioResource Center (BRC) through the National Bio-Resource Project of the MEXT, Japan. Human breast cancer cell line, MDA-MB-231, and human mammary epithelial cell line, MCF10A, were purchased from the American Type Culture Collection (ATCC). MCF-7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Nissui Pharmaceutical; Tokyo, Japan) supplemented with 10% inactivated fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA) in humidified atmosphere of 5% CO2 at 37 °C. MDA-MB-453 and MDA-MB-231 cells were maintained in Leibovitz-L15 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% inactivated FBS in humidified atmosphere of 0% CO2 at 37 °C. MCF10A cells were grown in mammary epithelial basal medium (MEBM; Lonza, Walkersville, MD, USA) supplemented with BPE, hydrocortisone, hEGF, insulin, gentamicin/amphotericin-B, and 100 ng/ml cholera toxin in humidified atmosphere of 5% CO2 at 37 °C. 293FT cell line was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and was maintained in DMEM supplemented with 10% inactivated FBS in humidified atmosphere of 5% CO2 at 37 °C.
5
Culturing HaCaT Keratinocytes in DMEM
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HaCaT keratinocytes were cultured in Dulbecco s modified Eagle medium (DMEM) ( Nissui, Tokyo, Japan) supplemented with 5％ fetal bovine serum (FBS) ( Biowest, Nuaillé, France) and incubated in a humidified atmosphere of 95％ air and 5％ CO 2 at 37℃.
6
Oxidative Stress Assay Reagents
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RK, pHPA, 2pHP-IA, pHPE, and H2O2 were purchased from Wako Pure Chemical Industries (Osaka, Japan). RD and EDTA were purchased from Nacalai Tesque (Kyoto, Japan). MEHQ was purchased from Tokyo Chemical Industry (Tokyo, Japan), respectively. Tyrosinase from mushroom, neutral red, and Triton X-100 reduced were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2′,7′-dichlorofluorescin diacetate (DCFDA) was purchased from EMD Millipore (Billerica, MA). Hoechst33342 was purchased from Life Technologies (Carlsbad, CA). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo). Dimethyl sulfoxide (DMSO) was purchased from Pierce Biotechnology (Rockford, IL). Ferrous sulfate was purchased from United States Pharmacopeial Convention (Rockville, MD). Dulbecco's modified Eagle medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo). Fetal bovine serum (FBS) was purchased from Biological Industries Israel (Kibbutz Beit-Haemek, Israel).
7
CRISPR Knockout Library Preparation
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The Human CRISPR Knockout Pooled Library (GeCKO v2) [17 (link)] comprising the lentiCas9‐Blast and lentiGuide‐Puro two vector system was purchased from Addgene (Watertown MA, USA, #1000000049) and prepared following the manufacturer's instructions. shRNA vectors (Table 2 ) were inserted into the CS‐Rfa‐CG backbone (provided by H. Miyoshi, RIKEN BioResource Research Center, Tsukuba, Japan). Lentiviruses were prepared via cotransfection of 1 × 107 HEK293FT cells per 10 cm culture plate with the appropriate vector along with the packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) in a 2 : 1 : 1 ratio (15 μg plasmid DNA per plate) using 60 μL of 1 mg·mL−1 polyethylenimine ‘MAX’ (Polysciences, Inc., Warrington PA, USA, #24765) in a 4 : 6 mix of Opti‐MEM reduced serum medium (Gibco, Waltham MA, USA, #22600050):Dulbecco's Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan, #5919). Lentiviral particles were collected via ultracentrifugation in an Optima XE‐90 Ultracentrifuge at an RCF of 76,800 x g (ravg) / 106,000 x g (rmax) 72 h post‐transfection and reconstituted in PBS for at least 24 h before use in downstream experiments.
8
Melanogenesis Regulation by α-MSH
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α-MSH was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Nissui Pharmaceutical (Tokyo, Japan). Antibodies against tyrosinase, TRP-1, TRP-2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MITF, phosphorylated CREB, and CREB were obtained from Cell Signaling Technology (Danvers, MA). Fetal bovine serum (FBS) was obtained from Gibco (Gaithersburg, MD, USA). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).
9
Cell Culture Protocols for Macrophages and Epithelial Cells
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Mouse macrophage RAW264.7 cells were cultured at 37°C under 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan), supplemented with 10% Fetal Calf Serum (FCS; JRH Biosciences, Lenexa, KS), 0.03% L-glutamine (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan), and antibiotic-antifungal combination agent (Gibco™ Antibiotic-Antimycotic; Thermo Fisher Scientific, Waltham, MA). Human alveolar basal epithelial A549 cells were cultures at 37°C under 5% CO2 in Ham’s F-12K medium (FUJIFILM Wako Pure Chemical Corporation), supplemented with 10% FCS, and antibiotic-antifungal combination agent.
10
Genetic Manipulation of HEK293A Cells
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HEK293A cells (Thermo Fisher Scientific) and their derivative G-protein-deficient HEK293A cells, including ∆Gs (GNAS/GNAL-deficient), ∆Gi (GNAI1/GNAI2/GNAI3/GNAO1/GNAT1/GNAT2/GNAZ-deficient), ∆Gq (GNAQ/GNA11-deficient), ∆G12 (GNA12/GNA13-deficient) and ∆ARRB (ARRB1/ARRB2-deficient)64 (link)–68 (link) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (GIBCO, Thermo Fisher Scientific) and penicillin-streptomycin-glutamine (complete DMEM). Transfection was performed by using a lipofection reagent, polyethylenimine (PEI) solution (Polyethylenimine “Max”, Polysciences). Typically, HEK293A cells were seeded in a 6-well culture plate at cell density of 2–3 × 105 cells per mL in 2 mL (per well hereafter) of the complete DMEM and cultured for one day in a humidified 37 °C incubator with 5% CO2. A PEI transfection solution was mixed by combining plasmid a solution diluted in 100 µL of Opti-MEM and 5 µL of 1 mg per mL PEI solution in 100 µL of Opti-MEM. The transfected cells were further incubated for one day before subjected to an assay as described below.
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