Human osteosarcoma cell line MG63, 143B, U2OS and Saos2 were purchased from Shanghai Institutes for Biological Sciences Cell Resource Center (China). The cell lines in this study were validated. The MG63 and 143B cell line were cultured in MEM medium (Gibco, USA) containing 10% FBS (Hyclone, USA) plus 1% penicillin and streptomycin (Solarbio, China). The U2OS and Saos2 cells were maintained in McCoy’s 5A medium (Hyclone) added with 10% FBS (Hyclone) plus 1% penicillin and streptomycin (Solarbio). All cells were placed in a 37 °C incubator with 5% CO2. The siRNA targeting circPIP5K1A (si-PIP5K1A), miR-515-5p mimics and inhibitor and the negative controls (NC), andYAP1 overexpression plasmid (pCMV-YAP1) were purchased from RiboBio (China). The MG63 and U2OS cell were seeded in 6-well plates, and cultured overnight to form a 60% monolayer, and transfected by the pCMV-YAP1 or RNA sequences through a lipofectamine 2000 (Invitrogen, USA) following manufactures’ description.
Penicillin and streptomycin
Manufactured by Solarbio
Sourced in China, United States, Israel
Penicillin and streptomycin are antibiotics commonly used in laboratory settings. Penicillin is a beta-lactam antibiotic that disrupts bacterial cell wall synthesis, while streptomycin is an aminoglycoside antibiotic that inhibits bacterial protein synthesis. These antibiotics are often used together to prevent bacterial contamination in various laboratory procedures and cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Sartorius (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Anti human cd3 antibody, by Thermo Fisher Scientific (3 mentions)
Anti human cd28 antibody, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Mem medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Solarbio (2 mentions)
Colo357, by Genechem (2 mentions)
Cf pac1, by Genechem (2 mentions)
Panc 1, by Genechem (2 mentions)
Sw1990, by Genechem (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Solarbio (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
P1400 100, by Solarbio (2 mentions)
72 protocols using penicillin and streptomycin
1
Osteosarcoma Cell Line Validation and Manipulation
2
Human and Murine Cancer Cell Culture
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HCT116 human colon cancer cells were obtained from the ATCC at the beginning of this project. Cells were maintained at 37 C under 5% CO 2 in McCoy's 5A (Cat. #GNM16600, Genom) with 10% (vol/vol) FBS (Cat. #10270, Gibco) supplemented with 1% (vol/ vol) penicillin and streptomycin (Cat. #P1400, Solarbio) and maintained in culture for a maximum of 2 months or 10 passages. Murine cell lines were also obtained from the ATCC at the beginning of our study and maintained in culture for a maximum of 2 months or 10 passages. CT26 murine colon carcinoma cancer cells and Raw264.7 murine macrophages were cultured at 37 C under 5% CO 2 in RPMI-1640 with 10% (vol/vol) FBS (Cat. #10270, Gibco) supplemented with 1% (vol/vol) penicillin and streptomycin (Cat. #P1400, Solarbio). All cells tested negative for Mycoplasma contamination and were authenticated on the basis of short tandem repeats fingerprinting before use.
3
Isolation and Culture of Rat Synovial Fibroblasts
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Synovial tissue specimens were obtained from rat ankle joints. Synovial cells (mainly fibroblast-like synoviocytes) were isolated from tissue explants and cultured as previously described [23 (link)]. Briefly, each sample was cut into small pieces and placed into a digestion solution containing collagenase (type I) (Sigma) for 1–2 h with brief mixing. After centrifugation, ethylenediaminetetraacetic acid (EDTA) (Solarbio) was added to the precipitate for digestion for 20−30 min, and the digestion was terminated when single or dispersed cell masses were observed under the microscope. Then, the cells were collected and cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone) containing 10% fetal bovine serum (FBS) (Sigma) and 1% streptomycin and penicillin (Solarbio) at 37°C with 5% CO2. After overnight incubation, nonadherent cells were removed, and adherent cells were seeded into 6-well culture plates (3 × 105 cells/ml/well).
All experiments were performed with cells obtained after passage 3. Cell-specific markers were used to identify synoviocyte populations. FLS cells were vimentin+ and CD68-, as demonstrated by specific fluorescence antibody staining (Abcam). FLS cells were treated with or without MSU (100 μg/ml) separately for 48 h. FLS cells and supernatants were harvested and frozen at -80°C for later cytokine analysis by qPCR and western blot.
All experiments were performed with cells obtained after passage 3. Cell-specific markers were used to identify synoviocyte populations. FLS cells were vimentin+ and CD68-, as demonstrated by specific fluorescence antibody staining (Abcam). FLS cells were treated with or without MSU (100 μg/ml) separately for 48 h. FLS cells and supernatants were harvested and frozen at -80°C for later cytokine analysis by qPCR and western blot.
4
Generating M1 and M2 Macrophages from Murine Bone Marrow
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The femur and tibia of C57BL/6 mice were used to isolate the bone marrow cells, which were then cultured for seven days in RPMI‐1640 medium (Life Technologies, UK) with 10% fetal bovine serum (FBS) (Gibco, USA), 1% streptomycin and penicillin (Solarbio, China), and 30% L929 culture supernatant medium to differentiate them into bone marrow derived macrophages (BMDM).28 Fresh media supplemented with the polarizing cytokines were added to modulate M1 or M2 macrophage polarization for further 2 days (M1: 100 ng/mL LPS; M2: 20 ng/mL IL‐4 and IL‐13).
Recombinant human M‐CSF (10 ng/mL × 5 days) and phorbol 12‐myristate 13‐acetate (50 ng/ml, PMA, Sigma‐Aldrich) for 2 days were utilized to stimulate CX3CR1+ monocytes and THP‐1 cells separately into macrophages. After treatment with DAC (5 μM)29 or phosphate buffer saline (PBS) (used as solvent control) during the differentiation period, IL‐4 and IL‐13 were added to induce the differentiation of M2 macrophages for 3 days. The purity and viability were examined by flow cytometry (Figure S1B,C ).
Recombinant human M‐CSF (10 ng/mL × 5 days) and phorbol 12‐myristate 13‐acetate (50 ng/ml, PMA, Sigma‐Aldrich) for 2 days were utilized to stimulate CX3CR1+ monocytes and THP‐1 cells separately into macrophages. After treatment with DAC (5 μM)
5
Culturing Human Thyroid Cell Lines
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Human ATC cell line FRO and human normal thyroid cell line Nthy-ori 3-1 were bought from Punosai (Wuhan, China). Cells were maintained in DMEM (Solarbio, Beijing, China) provided with 0.1% FBS (Gibco, Rockville, MD, USA) and 1% streptomycin and penicillin (Solarbio) and cultured at 37°C with 5% carbon dioxide (CO 2 ).
6
Antioxidant Compounds Characterization
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Folin-Ciocalteu’s phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ) and 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonicacid) (ABTS) were purchased from Sigma (St. Louis, MO); Calycosin (purity, ≥98%), formononetin (purity, ≥98%) were purchased from Mairier company (Shanghai, China); Ferulic acid (purity, ≥98%) and isoferulic acid (purity, ≥98%) were purchased from Yuanye Company (Shanghai, China); Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s minimum essential medium (DMEM) and Trypsin-EDTA (0.25% trypsin with EDTA-4Na) were purchased from Gibco (Grand Island, NY); The mixture of penicillin and streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Solarbio (Beijing, China); HPLC-grade acetonitrile was obtained from Fisher Scientific (Waltham, MA); Water was purified using a Milli-Q system from Millipore (Bedford, MA).
7
Osteogenic Differentiation of Third-Generation Cells
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The third-generation cells were placed in a six-well plate (2×105 cells/well). The medium was replaced with osteogenic induction medium, consisting of α-MEM supplemented with 10% fetal bovine serum (HyClone, United States), 1% penicillin and streptomycin (Solarbio, China), dexamethasone (10 nM, Solarbio), beta-Glycerol phosphate (10 mM, Solarbio) and ascorbic acid 2-phosphate (200 μM, Solarbio) when the cell density reached 80% of the plate. The medium was changed every 3 days. ALP staining (Meilunbio, China) was performed 7 days later to observe the early osteogenic differentiation. Moreover, after 14 days, ARS (Solarbio, China) was used to assess late osteogenic differentiation according to the manufacturer’s instructions. The staining was observed under a microscope at low magnification.
8
Impacts of ER Stress on Intestinal Lipid Metabolism
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Intestinal cells were isolated from healthy large yellow croaker and cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12) medium (Biological Industries, Israel) with 15% fetal bovine serum (FBS) (Biological Industries) and penicillin and streptomycin (Solarbio) in 5% CO2 atmosphere at 27°C. Intestinal cells were seeded into 6-well plates and cultured overnight. Cells were treated with 1 μM TM for different time points to explore the effects of ER stress on intestinal lipid metabolism and inflammation in vitro. Then cells were harvested for further analysis.
9
UPR Sensors Regulate Lipid and Inflammatory Genes
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The acc, scd1, dgat1, dgat2, il-1β, il-6, tnfα, and cox2 promoter fragments of large yellow croaker were cloned, and then they were constructed into the PGL3-basic vector to assemble reporter plasmids, respectively, using a ClonExpress II One Step Cloning Kit (Vazyme Biotech, China), according to previously described methods (26 (link)). The XBP1, CHOP, ATF4, and ATF6 CDS fragments were cloned and constructed into a PCS2+ vector to assemble expression plasmids, respectively. According to the manufacturer’s instructions, all plasmids for transfection were prepared by using the EasyPure HiPure Plasimid MinPrep Kit (TransGen Biotech, China).
HEK293T cells were cultured in DMEM high glucose medium (Biological Industries) with 10% FBS (Biological Industries) and penicillin and streptomycin (Solarbio) in 5% CO2 atmosphere at 37°C. To determine the effects of UPR sensors on the promoter activities of lipid synthesis and proinflammatory genes, HEK293T cells were cotransfected with reporter plasmid, expression plasmid, and phRL-CMV plasmid using Lipofectamine 2000 (Invitrogen, USA). After transfection for 24 h, cells were harvested and measured for luciferase activity using a Dual-Luciferase Reporter Assay Kit (TransGen Biotech), according to the manufacturer’s instructions.
HEK293T cells were cultured in DMEM high glucose medium (Biological Industries) with 10% FBS (Biological Industries) and penicillin and streptomycin (Solarbio) in 5% CO2 atmosphere at 37°C. To determine the effects of UPR sensors on the promoter activities of lipid synthesis and proinflammatory genes, HEK293T cells were cotransfected with reporter plasmid, expression plasmid, and phRL-CMV plasmid using Lipofectamine 2000 (Invitrogen, USA). After transfection for 24 h, cells were harvested and measured for luciferase activity using a Dual-Luciferase Reporter Assay Kit (TransGen Biotech), according to the manufacturer’s instructions.
10
Cell Culture of Pancreatic Cell Lines
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The human pancreatic cancer cell lines (PANC-1, SW1990, COLO357 and CF-PAC1) and the human pancreatic ductal cell line (HPDE) were purchased from GeneChem (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Solarbio, Beijing, China), and they were maintained in a 37 incubator containing 5% CO 2 . The medium was replaced every 24-48 h according to the cell density. Cells were observed under an inverted microscope and were digested with 0.25% trypsin (Gibco, Thermo Fisher Scientific, Waltham, MA,USA) to enable passaging of the cells when they reached 80% confluence.
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