293T (ATCC, USA) and 293T-hACE2 (Integral Molecular Inc., Philadelphia, PA, USA) were used for pseudo virus creation and testing. Both strains were grown in DMEM (Corning, USA) supplemented with heat inactivated 10% FCS (Sigma-Aldrich, Darmstadt, Germany) and 1% penicillin/streptomycin (Corning). 293 T-hACE2 cells were additionally supplemented with 10 mM HEPES (Corning) and 0,5% puromycin (LEXSY). Cells were grown at 37 °C in a humidified incubator (Sanyo) with 5% CO2. They were removed and split with accutase (Corning) followed by centrifugation at 4000 rpm for 5 min with a Megafuge 1.0R (Heraeus, Hanau, Germany). Cells were counted with an automatic cell counter (EVE, NanoEnTek) and plated out according to the test conditions.
Humidified incubator
Manufactured by Sanyo
Sourced in Japan, United States
The Sanyo Humidified Incubator is a laboratory equipment designed to maintain a controlled environment for cell culture and other biological applications. The device provides a consistent temperature and humidity level within the incubation chamber, creating an optimal environment for the growth and preservation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
293ft cell, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (2 mentions)
Penicillin, by Sangon (2 mentions)
Streptomycin, by Sangon (2 mentions)
Nci h1975, by National Collection of Authenticated Cell Cultures (2 mentions)
Mcf 7, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (2 mentions)
Rpmi 1640, by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
56 protocols using humidified incubator
1
Protocol for Pseudo Virus Creation and Testing
2
Transient Transfection of 293 Cells
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293FT cells (Invitrogen, Carlsbad, CA, USA) or 293CD4 cells (293 cells constitutively expressing human CD4) [37 (link)] were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were cultured in a 5% CO2 in a humidified incubator (Sanyo, Japan) and then transferred to 6- or 96-well plates 1 day before transfection, and Fugene HD (Fugene HD [µL]: DNA [µg]: DMEM [µL] = 5:2:200; Promega, Madison, WI, USA) was used for transient transfection. The transfection mix was incubated for 15 min at room temperature prior to addition to the cell culture in a drop-wise manner (10 µL/well). After indicated time after the transfection, the transfected cells were subjected to further analyses, as indicated below.
3
Microglia BV2 cell culture and JQ1 treatment
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Mouse microglia BV2 cells were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Biological Industries, Iseral) supplemented with 10% fetal bovine serum (FBS) (Lanzhou Bailing Biotechnology, China) and penicillin/streptomycin (BBI Life Sciences, China). The cells were maintained in a humidified incubator (Sanyo, Japan) with a 95% air/5% CO2 atmosphere at 37 °C. (+)-JQ1 (Nanjing Tianzhun, China) was dissolved in DMSO to a final concentration ranging from 0 to 10 μM. In the experiments indicated, the cells were pretreated with various concentrations of either (+)-JQ1 or DMSO 30 min prior to LPS (100 ng/ml) (Sigma-Alorich, USA)/Aβ (10 μM) (Sigma-Alorich, USA) treatment. Minomycine (2 μM) (Shyuanye, China) was administrated as the positive control. For NFκB signaling test, PDTC (p65 inhibitor), U0126 (ERK inhibitor) or SB203580 (p38 inhibitor) (Calbiochem, Germany) were treated with LPS or (+)-JQ1 for 24 h, followed by medium collection.
4
MCF-7 Cell Culture and Spheroid Formation
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MCF-7 cells were cultured in DMEM high glucose medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Bai Ling Biotechnology) and 1% penicillin-streptomycin (P/S) (Hyclone). MCF-7 CSCs were cultured in DME/F12 1:1 medium (Hyclone) supplemented with N2 (Gibco), B27 (Gibco), epidermal growth factor (EGF) (20 ng/mL, PeproTech), and basic fibroblast growth factor (bFGF) (10 ng/m, PeproTech). And the petri dishes used for CSCs were pre-coated with 1% Geltrex (Gibco) as described previously (Fu et al., 2017 (link)). All cells were cultured at 37°C under 5% CO2 in a humidified incubator (Sanyo).
5
Culturing Diverse Cell Lines
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The murine hepatoma cell line H22, human cervix epithelial carcinoma HeLa, and the human bronchial epithelial cell line BEAS-2B were obtained from the Shanghai Cell Center of Chinese Academy of Medical Science (Shanghai Institutes for Biological Sciences, Shanghai, People’s Republic of China). The H22 and HeLa cells were cultured with FA-free Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St Louis, MO, USA) with 1% penicillin and streptomycin, and 10% fetal bovine serum (FBS) (Gibco-BRL; Life Technologies, Carlsbad, CA, USA), while the BEAS-2B cells were incubated in serum-free medium; LHC-9 (Biofluids, Inc., Rockville, MD, USA) were incubated in a humidified incubator (SANYO Electric Co, Ltd, Moriguchi, Osaka, Japan) containing a 5% CO2-humidified atmosphere at 37°C.
6
Cell Culture of SH-SY5Y Neuroblastoma Cells
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The techniques that we used were according to the methods described previously by our experimental group [21 (link)]. SH-SY5Y cells were purchased from the Central Laboratory, the Affiliated Hospital of Qingdao University. Cells were cultured in DMEM/F12 (Gibco, USA) containing 2 mM L-glutamine, 10% FBS (Gibco, USA), 1% penicillin(100 U/ml of penicillin G), and 1% streptomycin (100 μg/ml of streptomycin). These cells were incubated with 5% CO2/95% air and maintained at 37°C in a humidified incubator (Sanyo, Osaka, Japan).
7
Phthalate Metabolism and Endocrine Disruption
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The MLTC-1 cell line was obtained from the Cell Institute of Shanghai (Shanghai, China) and cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 100 unit/mL penicillin, 100 unit/mL streptomycin and 10% (v/v) foetal bovine serum (Hyclone, USA). The cells were grown at 37 o C with 5% CO2 in a humidified incubator (SANYO, Japan). The MLTC-1 cells were seeded in 10-cm petri dishes and cultured for 48 h prior to further treatment. Phthalates were dissolved in DMSO. Cell viability was evaluated by the MTT proliferation assay (see SI Figure S1) to ensure that non-cytotoxic concentrations were employed for the following phthalates exposure experiments.
The first set of experiments was designed to assess the hydrolysis metabolism profiles of phthalate diesters and the different endocrine disrupting effects between After 48-h exposure, the culture medium was collected for phthalate metabolites analysis, and the cells were washed with 4 ml PBS and serum-free medium towards assessing steroidogenic functions. The cells were stimulated with hCG (0.1 U/ml) for 4 h in serum-free medium with 0.1% BSA. The medium was collected for 17-OHP, ASD and testosterone determination, and cells were either prepared for RNA extraction or gene expression analysis or for carboxylesterase activity determination.
The first set of experiments was designed to assess the hydrolysis metabolism profiles of phthalate diesters and the different endocrine disrupting effects between After 48-h exposure, the culture medium was collected for phthalate metabolites analysis, and the cells were washed with 4 ml PBS and serum-free medium towards assessing steroidogenic functions. The cells were stimulated with hCG (0.1 U/ml) for 4 h in serum-free medium with 0.1% BSA. The medium was collected for 17-OHP, ASD and testosterone determination, and cells were either prepared for RNA extraction or gene expression analysis or for carboxylesterase activity determination.
8
Culturing NSCLC Cell Lines
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NSCLC cell lines (A549 and NCI-H1975) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sangon, China). All cells were maintained in a humidified incubator (Sanyo, Japan) at 37°C and 5% CO2.
9
Culturing Rat H9c2 Cardiomyoblasts
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Rat embryonic cardiomyoblast-derived H9c2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 2 mM L-glutamine, 10% (v/v) FBS, and penicillin (100 U/mL)/streptomycin (100 μg/mL). Cells were maintained in a humidified incubator (Sanyo, Osaka, Japan) (95% air/5% CO2 at 37 °C) until 70–80% confluent prior to various experiments.
10
Culturing Human Glioblastoma U-87 MG Cells
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Human U-87 MG cells derived from human astrocyte glioblastoma grade III, were purchased from ATCC (HTB-14). Cells were cultured in a humidified incubator (Sanyo, Japan) at 37 °C in an atmosphere containing 5% of CO 2 in DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% of heat-inactivated FBS (fetal bovine serum), 1% antibiotic-antimycotic (streptomycine, amphotericin B, penicillin). Mycoplasma absence was confirmed using MycoAlert™ kit (Lonza, USA).
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