The B16-F10 mouse melanocyte cell line was kindly provided by Dr. Benilde Jiménez Cuenca, Instituto de Investigaciones Biomédicas «Alberto Sols» UAM-CSIC (Madrid, Spain). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin G (100 U/mL) and streptomycin (100 µg/mL) (HyClone Laboratories, South Logan, UT, USA). Cells were maintained under standard conditions at 37 °C, 5% humidity and 5% CO2 in an incubator (Heraeus HERAcell, Thermo Scientific, Waltham, MA, USA). B16-F10 cell line is a melanoma cell line and not normal melanocytes, but we consider that this cell line is appropriate for the present study. We consider that the use of a malignant cell line does not interfere with the parameters that have been evaluated in the study.
Penicillin g
Manufactured by Cytiva
Sourced in United States
Penicillin G is a pure, crystalline form of the antibiotic penicillin. It is used as a raw material in the production of various penicillin-based pharmaceutical products.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Cytiva (7 mentions)
Heraeus heracell, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Cytiva (3 mentions)
Rat bmscs, by Cyagen (1 mentions)
Low glucose dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Vero cell line atcc ccl81, by American Type Culture Collection (1 mentions)
Cercopithecus aethiops, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Thp 1 cells, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Cytiva (1 mentions)
L glutamine, by Cytiva (1 mentions)
Thp 1 cell, by American Type Culture Collection (1 mentions)
7 protocols using penicillin g
1
Culturing Murine Melanoma Cells for Research
2
Isolation and Culture of Human Dermal Fibroblasts
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Human dermal fibroblasts (HDF) were obtained from a skin biopsy after two rounds of trypsinization with 0.25% Trypsin-EDTA by shaking and at 37 °C. HDF were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin G (100 U/mL) and streptomycin (100 µg/mL) (HyClone Laboratories, South Logan, UT, USA). Cells were maintained at 37 °C, 5% humidity and 5% CO2 in an incubator (Heraeus HERAcell, Thermo Scientific, Waltham, MA, USA).
3
Characterization of Rat Bone Marrow Mesenchymal Stem Cells
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Rat BMSCs were purchased from Cyagen Biosciences (Guangzhou, China) and characterized by specific cell surface markers, including cluster of differentiation (CD)29, CD34, CD44, CD45, CD11b and CD90. The cells were highly positive for CD29 (83.99%), CD44 (99.69%) and CD90 (95.05%), and negative for CD34 (0.62%), CD45 (0.28%) and CD11b (4.25%), and were able to differentiate into osteoblasts, chondrocytes and adipocytes. The cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM; HyClone Laboratories, Inc., Logan, UT, USA) containing 10% fetal bovine serum (Hyclone Laboratories, Inc.), 10 U/ml penicillin G and 10 mg/ml streptomycin (Hyclone Laboratories, Inc.) in a 5% CO2 incubator at 37°C.
4
Cell Culture of Huh-7 and Vero Cells
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The Huh-7 hepatocarcinoma cell line was generously provided by Dr. Sonia Zúñiga (CNB-Spanish National Centre for Biotechnology, Madrid, Spain). This cell line was obtained from a liver tumor in a 57-year-old Japanese male and was used to test HCoV-229E infection [33 (link)]. The Vero cell line (ATCC-CCL81), which was derived from the kidney of an adult African green monkey (Cercopithecus aethiops) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in growth medium (GM) containing low-glucose Dulbecco’s modified Eagle medium (Life Technologies, Paisley, UK) supplemented with 5% fetal bovine serum (FBS), 1% (v/v) penicillin G (50 U/mL) and streptomycin (50 µg/mL) (HyClone Laboratories, Logan, UT, USA). Cells were maintained at 37 °C, in a humidified atmosphere of 5% CO2 (Heraeus HERAcell, Thermofisher. Waltham, MA, USA).
5
Culturing B16-F10 Melanocytes and Human Dermal Fibroblasts
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The B16-F10 mouse melanocyte cell line was kindly provided by Dr. Benilde Jiménez Cuenca, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC. Human dermal fibroblasts (HDF) were obtained from a skin biopsy by trypsinization with 0.25% Trypsin-Ethylenediaminetetraacetic acid (EDTA). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin G (100 U/mL), and streptomycin (100 µg/mL) (HyClone Laboratories, South Logan, UT, USA). Cells were maintained under standard conditions at 37 °C, 5% humidity, and 5% CO2 in an incubator (Heraeus HERAcell, Thermo Scientific, Waltham, MA, USA).
6
RAW264.7 Cell Culture Protocol
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The RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), being seeded in six-well plates with Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Life Technologies) and 100 U/mL penicillin G and 100 mg/mL streptomycin (HyClone Laboratories, Inc., Logan, UT, USA). The complete medium was free of mycoplasma. The cells were incubated at 37°C in a 5% CO2 incubator.
7
Monocyte Differentiation and Inflammatory Response
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THP-1 cells (American Type Culture Collection, Rockville, MD, USA) a human promonocytic cell lines were cultured in an RPMI 1640 medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10 IU/mL penicillin G, 10 μg/mL streptomycin, 2 mM L-glutamine, and 10% fetal calf serum (HyClone Laboratories, Logan, UT, USA). Briefly, THP-1 cells were differentiated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) for three days until >99% of cells were adherent. The cells were pretreated with BMS-345541 (25 μM), NF-κB by BAY 11-7082 (12 μM), or CAPE (20 μM) and tin-protoporphyrin (SnPP, the HO-1 inhibitor; 20 μM) at 37 °C for 1 h, and then treated with a pooled saliva sample obtained from patients with periodontitis for 24 h. All further steps were performed on ice or in a cold room unless otherwise noted.
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