Mouse bone marrow–derived MSCs were isolated and cultured as described previously [28 ]. Briefly, bone marrow was harvested via repeated washing of the tibia cavity; then, the aspirate was centrifuged at 1000 rpm for 5 min. The cell containing pellets were resuspended in DMEM/F12 (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, USA), plated on 6-well cell culture clusters, and cultured at 37 °C in a 5% CO2 constant-temperature incubator (Thermo, USA). Coronary endothelial cells (CAECs) were purchased from the Procel life science Laboratory (Wuhan, China), and cultured in endothelial cell medium (Sciencell, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, USA) at 37 °C in a 5% CO2 constant-temperature incubator (Thermo, USA).
Co2 constant temperature incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CO2 constant temperature incubator is a laboratory equipment designed to maintain a controlled environment for cell culture and other biological applications. The core function of this incubator is to provide a stable temperature and precise CO2 levels within the interior chamber to support the optimal growth and maintenance of cell lines and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Endothelial cell medium (ecm), by ScienCell (2 mentions)
H1975, by American Type Culture Collection (1 mentions)
H1650, by American Type Culture Collection (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Ab219800, by Abcam (1 mentions)
Ab214185, by Abcam (1 mentions)
Empagliflozin, by MedChemExpress (1 mentions)
Ab138483, by Abcam (1 mentions)
Goat anti rabbit igg h l, by Beyotime (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Biotin labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Tmb chromogen solution, by Beyotime (1 mentions)
Cy3 labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
5 protocols using co2 constant temperature incubator
1
Isolation and Culture of Mouse Bone Marrow-Derived MSCs and Coronary Endothelial Cells
2
Culturing Lung Cell Lines
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Normal human lung epithelial cells BEAS-2B and lung adenocarcinoma cell line H1650, H1975, A549, and H292 were purchased from American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). All cell lines were incubated in Roswell Park Memorial Institute (RPMI)-1640 culture medium containing 10% inactivated FBS (Gibco Company, Grand Island, N.Y., U.S.A.), 100 units/ml penicillin, and 100 mg/ml streptomycin (HyClone Company, Logan, UT, U.S.A.) in a 5% CO2 constant temperature incubator (Thermo Fisher Scientific, Carlsbad, CA, U.S.A.) at 37°C. When the cells confluence reached 80%, the cells were detached using 0.25% trypsin for subsequent experiments.
3
Cell Culture Protocol for CRC Lines
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The human CRC cell lines SW480 and RKO were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle medium (Gibco, USA) supplemented with 10% fetal bovine serum (BI, USA) and 100 IU/mL penicillin. All cells were incubated in a CO2 constant temperature incubator (37°C, 21% O2, and 5% CO2) (Thermo Fisher Scientific, Rockford, IL, USA) for normal oxygen culture or in a hypoxic incubator (37°C, 1% O2, and 5% CO2) for hypoxic culture for 24 h.
4
Molecular Pathways of GSDMD-Mediated Inflammation
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Dimethyl sulfoxide (DMSO) (D8371, Solaibao, Beijing, China); Cell Counting Kit-8 (CA1210, Solaibao); BCA Protein Concentration Determination Kit (P0012S, Beyotime, Shanghai, China); RIPA buffer (R0010, Solaibao); Hematoxylin and Eosin Staining Kit (C0105S, Beyotime); 4% paraformaldehyde (BL539A, Biosharp, Hefei, China); 10% neutral buffer formalin fixative (Thermo right, Changchun, China); TMB Chromogen Solution (P0211, Beyotime); DAPI (C0065, Solaibao); Anti-GSDMD antibodies (ab219800, Abcam, Cambridge, UK); Anti-NLRP3 antibodies (ab214185, Abcam); Anti-caspase-1 antibodies (ab138483, Abcam); β-actin polyclonal antibody (20,536-1-AP, Proteintech, Rosemont, IL, USA); Anti-caspase-1 antibody (AB1871, Sigma,-Aldrich, USA); NLRP3 recombinant rabbit monoclonal antibody (sc06-23, Invitrogen, Waltham, MA, USA); Cy3-labeled goat anti-rabbit IgG (H + L) (A0516, Beyotime); Goat-anti-rabbit IgG (H + L) (A0208, Beyotime); Biotin-labeled Goat Anti-Rabbit IgG(H + L) (A0277, Beyotime); Empagliflozin (HY-15409, MedChemExpress, Princeton, NJ, USA); ECL Kit (Millipore, Billerica, MA, USA). CO2 constant temperature incubator (Thermo Fisher, USA); fluorescence microscope (DM500, Leica, Germany); Electrophoresis instrument electrophoresis system (Bio-Rad, Hercules, CA, USA); ChemiDoc™MP system (Bio-Rad).
5
Murine Cell Culture and Analysis
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Materials: DMEM (Hyclone). Fetal bovine serum (Gibco). PBS, 0.25% trypsin (Hyclone). RIPA lysate (Beijing Solebo). SYBR green PCR kit (Thermo); Trizol (invitrogen). BCA Protein Quantitation Kit (Thermo). Tris-HCl, pH = 8.8 electrophoretic buffer solution (Beijing Solebo). CO2 constant temperature incubator (Thermo). Real-time detector (ABI) (ABI-7300). Flow cytometry (Accuri C6, BD). Refrigerated centrifuge (TG-16M) (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.). Consumables for cell culture (Corning). C57BL/6J SPF level mice (4 week, 13-16 g) were purchased form Weitong Lihua Laboratory Animal Technology Co., Ltd. All animals were maintained on an ad libitum diet of laboratory chow and water and approved by the × × University of Animal Care Committee.
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