Human breast cancer cell lines, MCF-7 (an estrogen and progesterone receptor positive, hormone responsive cell line [43 (link)]) and MDA-MB-231 (a triple receptor negative cell line [46 (link)]) were cultured in Dulbecco’s Modified Eagles Medium (DMEM)/high glucose medium, supplemented with 10% fetal bovine serum (FBS), 10,000 units/mL penicillin/streptomycin (Pen/Strep), and 50 µg/mL gentamicin, while the human cervical cancer cell line, HeLa [44 (link)] was grown in DMEM/high glucose medium supplemented with 7% fetal calf serum (FCS), 10,000 units/mL Pen/Strep, and gentamicin (all reagents by HyClone Laboratories, Inc., Logan, UT, USA). The normal breast cancer epithelial cell line, MCF-10A [45 (link)] was cultured in DMEM/F12 medium (DMEM/Hams nutrient mixture) and 5% horse serum supplemented with 10,000 units/mL of Pen/Strep, 10 μg/mL insulin, 20 ng/mL epidermal growth factor (EGF), 0.5 mg/mL hydrocortisone, and 100 ng/mL cholera toxin (all reagents by Sigma-Aldrich, St. Louis, MO, USA). The cell lines were maintained at 37 °C in a 5% CO2 humidified incubator.
Gentamicin
Manufactured by Cytiva
Sourced in United States
Gentamicin is a broad-spectrum antibiotic used in various laboratory applications. It is effective against a wide range of gram-positive and gram-negative bacteria. Gentamicin is commonly used in cell culture media to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o, by Merck Group (5 mentions)
Dulbecco s modified eagle s medium dmem, by Cytiva (2 mentions)
Recombinant human il 1β, by R&D Systems (2 mentions)
Recombinant human interleukin 1β il 1β, by R&D Systems (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt assay, by Merck Group (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
Icam 1, by Abcam (1 mentions)
Mcp 1, by R&D Systems (1 mentions)
25 cm2 flask, by Greiner (1 mentions)
Gentamicin, by American Type Culture Collection (1 mentions)
24 well tissue culture plate, by BD (1 mentions)
Phosphorylated p38, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Recombinant human interleukin il 1β, by R&D Systems (1 mentions)
Phosphorylated nf κb, by Cell Signaling Technology (1 mentions)
Phosphorylated erk, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
8 protocols using gentamicin
1
Culturing Diverse Breast and Cervical Cancer Cell Lines
2
Western Blot Analysis of Fibrosis Markers
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Antibodies for periostin, β-actin, fibronectin, collagen Iα, α-smooth muscle actin (SMA), phosphorylated (p)- and total (t)- extracellular signal-related kinase (ERK), p-p38, t-p38, p-SMAD 1/5/8, t-SMAD1/5/8, p-SMAD2, t-SMAD2, interleukin (IL)-8, IL-6, monocyte chemoattractant protein-1 (MCP-1), p-nuclear factor (NF)-κB, t-NF-κB, p-Akt, t-Akt, peroxisome proliferator activator gamma (PPARγ), CCAAT-enhancer-binding protein (C/EBP)α and β used in the study are listed in detail in Supplementary Table 1
3
Characterizing PCSK9 Regulation in Cells
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The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling Technology (Beverly, MA, USA), Novus Biologicals (Centennial, CO, USA), and Abcam (Cambridge, UK). The antibodies used in the study are listed in detail in Supplementary Table 1
4
Cytokine Response Assay in Cell Cultures
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The antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), BD Biosciences (San Jose, CA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Novus Biologicals (Centennial, CO, USA), and Abcam (Cambridge, UK). The antibodies used are listed in detail in Supplementary Table S1 . CHIR 99021 was a product of Tocris Bioscience (Minneapolis, MN, USA). Recombinant human IL-1β was a product from R&D Systems (Minneapolis, MN, USA). The enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and IL-8 were purchased from R&D Systems (Minneapolis); for MCP-1, from R&D Systems (Abingdon, UK); and for ICAM-1, from Abcam. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and gentamicin were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA).
5
Cell Culture and Invasion Assay Protocol
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Human epithelium cells (INT407, ATCC CCL-6) were purchased from ATCC and cultured at standard condition (37°C, 5% CO2, 95% humidity) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 100 μg/mL gentamicin (HyClone Laboratories Inc., Logan, UT, United States). The cultured cells were seeded at approximately 2 × 105 cells/mL/well into 24-well tissue culture plates (BD Falcon, Franklin Lakes, NJ, United States) to reach 80–90% confluence monolayer at standard condition for cell adhesion assay. The post-confluent INT-407 cell monolayers were rinsed with PBS and stabilized in antibiotic-free DMEM for 1 h prior to the invasion assay.
Human macrophage cell line (U937, ATCC CRL3253) was purchased from ATCC and grown at standard condition in RPMI-1640 Medium supplemented with 10% FBS and 100 μg/mL gentamicin. An aliquot of 6 mL cell suspension containing 1 × 106 cells were transferred into 25 cm2 flask (Greiner Bio-One, Monroe, NC, United States) and cultured at standard condition for 24–30 h. After time, the cell monolayer was washed for three times with RPMI for further bacterial infection.
Human macrophage cell line (U937, ATCC CRL3253) was purchased from ATCC and grown at standard condition in RPMI-1640 Medium supplemented with 10% FBS and 100 μg/mL gentamicin. An aliquot of 6 mL cell suspension containing 1 × 106 cells were transferred into 25 cm2 flask (Greiner Bio-One, Monroe, NC, United States) and cultured at standard condition for 24–30 h. After time, the cell monolayer was washed for three times with RPMI for further bacterial infection.
6
Analysis of Adipocyte Differentiation
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E-GS was obtained from Sigma–Aldrich, Inc. (St. Louis, MO, USA). Oil Red O was purchased from Sigma–Aldrich, Inc. DMEM, penicillin, gentamicin, and FBS were acquired from Hyclone Laboratories, Inc. (Logan, UT, USA). Recombinant human interleukin (IL)-1β was obtained from R&D Systems (Minneapolis, MN, USA). Anti-CCAAT-enhancer-binding protein (C/EBP) α and β, anti-sterol regulatory element-binding protein-1 (SREBP-1), anti-peroxisome proliferator activator gamma (PPARγ), and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated ERK, Akt, phosphorylated Akt, c-Jun NH(2)-terminal kinase (JNK), phosphorylated JNK, NF-κB, phosphorylated NF-κB, p38, and phosphorylated p38 were all purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphorylated levels of proteins were normalized to their respective total protein levels for each phosphorylation analysis.
7
Cigarette Smoke-Induced Cell Viability Assay
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and gentamicin were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Caffeine was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS) assay solution was purchased from Promega Corporation (Madison, WI, USA). Cigarette smoke extract (CSE) was freshly prepared within an hour of each experiment from commercially available filtered cigarettes [Marlboro 20 class A cigarettes (8.0 mg tar; 0.7 mg nicotine); Philip Morris Korea, Inc., Seoul, Korea] as described in our previous study [12] . Recombinant human IL-1β was purchased from R&D Systems (Minneapolis, MN, USA) and Oil Red O was purchased from Sigma-Aldrich Corp.
8
Fibrosis Regulation by TGF-β Signaling
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and gentamicin were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). DMEM/F-12 and penicillin-streptomycin were obtained from Welgene, Inc. (Daegu, Korea). Recombinant transforming growth factor (TGF)-β was supplied by R&D Systems (Minneapolis, UT, USA). ERN1 A small-interfering RNA (siRNA) against IRE1α (#AM51331) and a negative-control siRNA (#4390843) were purchased from Ambion/Applied Biosystems (Austin, TX, USA). Bafilomycin A1 (Baf-A1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against IRE1a, protein kinase B (AKT), phosphorylated AKT, extracellular signal-regulated kinase (ERK), p-ERK, p38, p-p38, total SMAD family member 2 (t-SMAD2), p-SMAD2, t-SMAD3, p-SMAD3, t-SMAD4, p-SMAD4, light chain (LC) 3B-I/II, autophagy-related (ATG) 5, and ATG7 were obtained from Cell Signaling Technology (Beverly, MA, USA). Fibronectin and an anti-p62 antibody were supplied by Becton Dickinson (Franklin Lakes, NY, USA). An α-smooth muscle actin antibody (SMA) antibody from Sigma-Aldrich. An p-IRE1α antibody was supplied by Invitrogen (Carlsbad, CA, USA). A collagen 1α antibody was supplied by Abcam (Cambridge, MA, USA). A β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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