Experiments were performed using two human cell lines, namely epithelial human lung adenocarcinoma A549 (ATCC CCL-185) and human embryonic kidney HEK293 (ATCC CRL-1573). Both cell lines adhere to plastic and glass surfaces and were maintained in the cell culture under the same conditions. Cells were grown in Dulbecco Modified Eagle’s Medium (DMEM, Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% of foetal bovine serum (FBS, Sigma Aldrich, USA), and incubated in the cell incubator (Thermo Fischer Scientific, Waltham, MA, USA) at 37 °C and 5% CO2 in a humidified atmosphere.
Cell incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
A cell incubator is a laboratory equipment used to maintain optimal environmental conditions for cell cultures. It provides a controlled temperature, humidity, and atmospheric composition (typically containing 5-10% CO2) to support the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (35 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Rpmi 1640, by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Bxpc 3, by American Type Culture Collection (3 mentions)
Aspc 1, by American Type Culture Collection (3 mentions)
Penicillin streptomycin, by Solarbio (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Sartorius (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Mia paca 2, by American Type Culture Collection (2 mentions)
Panc 1, by American Type Culture Collection (2 mentions)
Bxpc 3, by Thermo Fisher Scientific (2 mentions)
Aspc 1, by Thermo Fisher Scientific (2 mentions)
Incubator chamber, by Embrient Inc (2 mentions)
M3 base, by INCELL (2 mentions)
98 protocols using cell incubator
1
Cell Culture Protocol for A549 and HEK293
2
Vascular cell culture protocol
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The human aortic vascular smooth muscle cell line (T/G HA-VSMC), which was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), was a gift by Dr. Chen Jian’s Lab. The human umbilical vein endothelial cell line (PUMC-HUVEC-T1) and human monocytic cell line (THP-1) were purchased from National Infrastructure of Cell Line Resource of China (Beijing, China). T/G HA-VSMC and PUMC-HUVEC-T1 cell lines were maintained in high-glucose DMEM (Life Technologies, Gibco) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Gibco), MEM Non-essential amino acids (MEM NEAA 100×, Life Technologies, Gibco), penicillin (100 U mL−1, Gibco, USA) and streptomycin (100 μg mL−1, Gibco, USA). THP-1 cells were maintained in RPMI 1640 Medium (Caisson Labs) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL−1) and streptomycin (100 μg mL−1). The cell lines were cultured in cell incubator (37 °C, 5% CO2) (Thermo Fisher Scientific). Human DiI-Acetylated LDL and LDL were purchased from Yeasen (Shanghai, China).
3
NHDF Culture and Compound Screening
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NHDFs (juvenile foreskin, cryopreserved, 5 × 105 cells) were obtained from PromoCell (Sickingenstr, Heidelberg, Germany) and stored in a nitrogen tank. NHDFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning, Manassas, VA, USA, pH 7.4) containing 10% fetal bovine serum (FBS; Atlas, Fort Collins, CO, USA) and 100 U/mL penicillin–streptomycin (Gibco, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO2 at 37 °C in a cell incubator (Thermo Scientific, Waltham, MA, USA). The isolated compounds were dissolved in dimethyl sulfoxide (Biosesang, Seong-Nam, Korea) to prepare 10 mM stock solutions. The isolated compounds were diluted in DMEM without FBS at concentrations of 25, 50, and 100 μM. The concentration of the TNF-α stock solution was 20 μg/mL, and the final treatment concentration was 20 ng/mL. In all experiments, the cells were seeded onto a cell culture plate and starved in DMEM without FBS overnight. The cells were then co-treated with TNF-α and the isolated compounds.
4
Culturing Pancreatic Cell Lines
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As described in previous studies [19 (link),20 ], the human ductal cell line (hTERT-HPNE) and pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, PANC-1, T3M4, and BxPC-3 were bought from ATCC. Pancreatic cancer line PaTu8988 was provided from PharmLab (PharmLab, Beijing, China). MIA PaCa-2, PANC-1 (DMEM, Gibco, Cat. no. C11995500), AsPC-1, BxPC-3, and T3M4 (RPMI 1640, Gibco, Cat. no. C11875500) were cultured in cell culture dishes (NEST Biotechnology, Wuxi, China) in a humidified incubator at 37 °C with 5% CO2. The hTERT-HPNE was cultured in 75% DMEM without glucose and 25% M3 Base (Incell, Cat. no. M300F-500) supplemented with 10 ng/mL human recombinant EGF (CST, Cat. no. 72528), 5.5 mM D-glucose (1 g/L), 5% FBS, and 0.75 mg/mL puromycin (MCE, Cat. no. 58-58-2). All cell lines were authentic by short tandem repeats profile. Cells were maintained in a cell incubator (ThermoFisher, Waltham, MA, USA) with 5% CO2 and 20% O2 for a normoxic condition and incubator chamber (Billups-rothenberg, San Diego, CA, USA) flushed with 5% CO2 and 95% N2 for hypoxic condition.
5
Rheumatoid Arthritis Fibroblast-like Synoviocyte Cell Treatment
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Human rheumatoid arthritis fibroblast-like synoviocyte cell line MH7A was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). MH7A cells were cultured in the DMEM/high glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA). Cells were incubated in the cell incubator (Thermo, USA) at 37°C under 5% CO2.
Cells were divided into 4 groups. The blank control cells (BC) were cultured as normal. The negative control cells (NC) were cultured in the medium containing 20% normal serum. The JWFSN-treament cells (JWFSN) were cultured in the medium with 20% JWFSN-containing serum. The kartogenin + JWFSN treament cells (KGN + JWFSN) were pretreated using 10−6 M kartogenin [19 (link)] for 2 h and then cultured in the medium with 20% JWFSN-containing serum.
Cells were divided into 4 groups. The blank control cells (BC) were cultured as normal. The negative control cells (NC) were cultured in the medium containing 20% normal serum. The JWFSN-treament cells (JWFSN) were cultured in the medium with 20% JWFSN-containing serum. The kartogenin + JWFSN treament cells (KGN + JWFSN) were pretreated using 10−6 M kartogenin [19 (link)] for 2 h and then cultured in the medium with 20% JWFSN-containing serum.
6
Cultivation of Pancreatic Cell Lines
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Human pancreatic cancer cell lines, including AsPC-1, BxPC-3, MIA PaCa-2, PANC-1, T3M4 and normal pancreatic ductal cell (hTERT-HPNE) were purchased from the American Type Culture Collection (ATCC). Human pancreatic cancer cell line PaTu8988 was from DSMZ. Human umbilical vein endothelial cell (HUVEC) was purchased from the Pharmlab in china. The HEK-293T was generously provided by professor Mao in the department of Biochemistry and Molecular Biology in Peking university. All cell lines were authenticated by short tandem repeats (STR).
The hTERT-HPNE was cultured in in 75% DMEM without glucose (Gibco, USA) and 25% M3 Base (Incell, USA) supplemented with 10 ng/ml human recombinant EGF (CST, USA), 5.5 mM D-glucose (1 g/L), 5% fetal bovine serum (Gibco, USA), 0.75 mg/ml puromycin (sigma, USA). AsPC-1, BxPC-3, T3M4 were cultured in RPMI 1640 (Gibco, USA). MIA PaCa-2, PANC-1, PaTu8988, HEK-293T were cultured in DMEM. HUVEC was cultured in Endothelial Cell Medium (ScienCell, USA) supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 1% penicillin-streptomycin solution (KeyGEN, china). Cells were maintained in cell incubator (ThermoFisher, USA) with 5% CO2 and 20% O2 for normoxic condition and incubator chamber (Billups-rothenberg, USA) flushed with 5% CO2 and 95% N2 for hypoxic condition.
The hTERT-HPNE was cultured in in 75% DMEM without glucose (Gibco, USA) and 25% M3 Base (Incell, USA) supplemented with 10 ng/ml human recombinant EGF (CST, USA), 5.5 mM D-glucose (1 g/L), 5% fetal bovine serum (Gibco, USA), 0.75 mg/ml puromycin (sigma, USA). AsPC-1, BxPC-3, T3M4 were cultured in RPMI 1640 (Gibco, USA). MIA PaCa-2, PANC-1, PaTu8988, HEK-293T were cultured in DMEM. HUVEC was cultured in Endothelial Cell Medium (ScienCell, USA) supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 1% penicillin-streptomycin solution (KeyGEN, china). Cells were maintained in cell incubator (ThermoFisher, USA) with 5% CO2 and 20% O2 for normoxic condition and incubator chamber (Billups-rothenberg, USA) flushed with 5% CO2 and 95% N2 for hypoxic condition.
7
Articular Chondrocyte In Vitro Culture
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In this study, we chose mice articular chondrocytes (CP-M092, Procell, Wuhan, China) for in vitro experiments. The above articular chondrocytes were cultured with complete culture medium of mice articular chondrocytes (CM-M092, Procell) and stored in cell incubator (51032124, Thermo Fisher Scientific, MA, USA), growth conditions: 37°C, 5% CO2. The above chondrocytes were cultured in 24-well plates for 24 h.
8
Transfection of BJ Cells for miR-27b Study
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BJ cells (CRL-2522; American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) and supplemented with 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA). Cells were incubated in a cell incubator (Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2 and saturated humidity.
BJ cells in the logarithmic phase were seeded into 6-well plates (2x105/well) and inoculated for 24 h. The wells were divided into the control, miR-27b, MC, inhibitor and IC groups and Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect corresponding reagents into cells for 12 h to construct BJ cells exhibiting miR-27b overexpression or inhibition.
BJ cells in the logarithmic phase were seeded into 6-well plates (2x105/well) and inoculated for 24 h. The wells were divided into the control, miR-27b, MC, inhibitor and IC groups and Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect corresponding reagents into cells for 12 h to construct BJ cells exhibiting miR-27b overexpression or inhibition.
9
Culturing Human Vascular Smooth Muscle Cells
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HVSMCs (C1601) ordered from WHELAB (Shanghai, China) were incubated with complete Dulbecco’s modified eagle medium (DMEM) (M1023, WHELAB, China) in a cell incubator (3111, THERMO, MA, USA).
10
Culturing HEK293T Cells and Cortical Neurons
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HEK293T cells (CRL-11268, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin–streptomycin (100 μg/ml and 100 μg/ml).
Cortical neurons were obtained from postnatal day 0 (P0) pups of Kunming mice, as described previously (Wang et al., 2020 (link)). Briefly, the cortex was dissected from newborn mice, digested to single cell with 0.25% trypsin (Gibco) for 12 min at 37°C. Then cells were plated at a density of 80,000 per circular glass coverslips (12 mm diameter) coated with poly-L-lysine (Sigma). Cells seeding medium contains MEM (Gibco), 2% (v/v) B27 (Gibco), 0.5% (w/v) glucose, 100 mg/L transferrin (Sigma), 5% (v/v) fetal bovine serum (Gibco), and 2 mM Ara-C (Sigma). During the process of neuron culture, the growth medium was needs to replace in DIV1 (the day 1 in vitro), DIV4, and DIV9. Each time 500 μl medium was taken out and 600 μl was added in. Different concentrations (0 μM, 10 μM, or 50 μM) of Zn2+ were added in culture medium from DIV1.
HEK293T cells and neurons were all grown at 37°C, 5% CO2 in a cell incubator (Thermo).
All animal procedures were carried out in accordance with the animal use rules of South-Central Minzu University and the requisite approvals of the animal use committee.
Cortical neurons were obtained from postnatal day 0 (P0) pups of Kunming mice, as described previously (Wang et al., 2020 (link)). Briefly, the cortex was dissected from newborn mice, digested to single cell with 0.25% trypsin (Gibco) for 12 min at 37°C. Then cells were plated at a density of 80,000 per circular glass coverslips (12 mm diameter) coated with poly-L-lysine (Sigma). Cells seeding medium contains MEM (Gibco), 2% (v/v) B27 (Gibco), 0.5% (w/v) glucose, 100 mg/L transferrin (Sigma), 5% (v/v) fetal bovine serum (Gibco), and 2 mM Ara-C (Sigma). During the process of neuron culture, the growth medium was needs to replace in DIV1 (the day 1 in vitro), DIV4, and DIV9. Each time 500 μl medium was taken out and 600 μl was added in. Different concentrations (0 μM, 10 μM, or 50 μM) of Zn2+ were added in culture medium from DIV1.
HEK293T cells and neurons were all grown at 37°C, 5% CO2 in a cell incubator (Thermo).
All animal procedures were carried out in accordance with the animal use rules of South-Central Minzu University and the requisite approvals of the animal use committee.
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