Mouse skeletal muscle cells, C2C12 myoblasts, were purchased from the Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). Cells were maintained in 90% Dulbecco’s modified Eagle’s medium (DMEM; 11965; Gibco, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (10437; Gibco, Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin-amphotericin B (P/S/A; Biological industries, Kibbutz, Beit HaEmek, Israel) at 37°C in a 5% CO2 atmosphere. Differentiation was induced by changing the medium to DMEM containing 2% horse serum (HS) and 1% P/S/A when the cells attained 90% confluence [9 (link),22 (link)]. The myotubes matured to striated cells by the fifth day after sowing, and the cultures were used for experiments as described previously [23 (link)]. The myotubes were assigned to 3 groups to investigate the effects of AS on myotubes: (1) non-AS supplement (normal growth medium: 2%HS/DMEM; NON); (2) IGF-1 supplement (10 ng/mL, in 2% HS/DMEM; as a positive control; IGF-1); and (3) AS supplement (10 ng/mL, in 2% HS/DMEM; AS).
Penicillin streptomycin amphotericin b p s a
Manufactured by Sartorius
Sourced in United States
Penicillin-streptomycin-amphotericin B (P/S/A) is a solution containing a combination of antibiotics and an antifungal agent commonly used in cell culture applications. It serves as a broad-spectrum antimicrobial supplement to prevent bacterial and fungal contamination in cultured cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Inositol, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Balb 3t3 cells, by RIKEN BioResource Center (1 mentions)
Alpha minimum essential medium α mem, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Folic acid, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (1 mentions)
1 2 dilauroyl sn glycero 3 phosphocholine dlpc, by Avanti Polar Lipids (1 mentions)
Hek293t cells, by RIKEN BioResource Center (1 mentions)
Ham s f 12 nutrient mixture dmem f12, by HiMedia (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dmem f12, by Sartorius (1 mentions)
Trypsin, by Merck Group (1 mentions)
5 protocols using penicillin streptomycin amphotericin b p s a
1
C2C12 Myoblast Differentiation and Supplementation
2
Cell Culture and Lipid Nanoparticle Preparation
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Human embryonic kidney (HEK) 293T cells, mouse melanoma B16-F10 cells, and mouse embryonic fibroblast BALB/3T3 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu City, Taiwan, ROC). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Gaithersburg, MD, USA) supplemented with inactivated 10% foetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin amphotericin B (PSA; Biological industries, New York, NY, USA). Human natural killer NK-92 MI cells were grown in alpha minimum essential medium (αMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 12.5% horse serum (Gibco BRL, Gaithersburg, MD, USA) and 12.5% FBS. All cells were incubated at 37 °C in an atmosphere of 5% CO2.
Male C57BL/6 mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (NLAC, Taipei City, Taiwan, ROC).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Avanti Polar Lipids, Inc.) and polyethylene glycol (PEG, MW 1.500) (Shimo-Meguro, Meguro-Ku, Tokyo, Japan), PEG (MW 8.000, Sigma-Aldrich) and polyethylenimine (PEI, branched, MW 25.000, Sigma-Aldrich) were purchased to prepare LPPC.
Male C57BL/6 mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (NLAC, Taipei City, Taiwan, ROC).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Avanti Polar Lipids, Inc.) and polyethylene glycol (PEG, MW 1.500) (Shimo-Meguro, Meguro-Ku, Tokyo, Japan), PEG (MW 8.000, Sigma-Aldrich) and polyethylenimine (PEI, branched, MW 25.000, Sigma-Aldrich) were purchased to prepare LPPC.
3
Hematoxylin Staining of HA22T/VGH Cells
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HA22T/VGH (HA22T, #60168) cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and maintained in a 3:2 mixture of Dulbecco’s modified Eagle medium and Ham’s F-12 Nutrient Mixture (DMEM/F12, 3:2; HIMEDIA, Mumbai, India) supplemented with 8% fetal bovine serum (FBS; ThermoFisher, Waltham, MA, USA) and 1% penicillin-streptomycin-amphotericin B (P/S/A; #03-033-1B, Biological Industries, Beit-Haemek, Israel). Cultures were grown in a 37 °C incubator with an atmosphere containing 5% CO2. Hematoxylin staining was utilized to visualize the cell nucleus. The treated cells were fixed with 4% paraformaldehyde (PFA) for 10 min and stained with hematoxylin (#GHS3, Sigma-Aldrich, St. Louis, MO, USA) for 5 min.
4
Cultivation of Human OSCC Cell Lines
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The human OSCC cell lines, OE and SAS, were grown in RPMI medium (GIBCO, Eggenstein, Germany) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Eggenstein, Germany) and 1% Penicillin-Streptomycin-Amphotericin B (PSA) (Biological industries, Cromwell, CT, USA). The OE (OECM-1) cells were obtained from Merck Millipore, Darmstadt, Germany. The SAS cells were kindly provided by Dr. Michael Hsiao at Genomics Research Center, Academia Sinica, Taipei, Taiwan.
5
Osteoarthritic Chondrocyte Culture and Nicotine-Induced OA Model
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The human chondrocytes cultures were obtained from patients who underwent joint
replacement therapy. The osteoarthritic cartilage harvested from patients was
turned into pieces and digested with an enzymatic solution [8 mg/ml
hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), 8 mg/ml collagenase
(Sigma-Aldrich), and 2.5 mg/ml trypsin (Sigma-Aldrich)] for 6 h at 37°C. The
cellular suspension was centrifuged at 1,500 rpm for 5 min and resuspended into
Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, New York, NY, USA)
with 10% fetal bovine serum (FBS; Gibco BRL), and 1%
Penicillin-Streptomycin-Amphotericin B (PSA) (Biological Industries, Beit
Haemek, Israel) in a humidified atmosphere containing 5% CO2.
Thereafter, the primary OA chondrocytes were cultured, passaged, and maintained
in DMEM/F12 medium.
Based on previous reports showing various inhibitory activities of nicotine
against bone density19 (link), human renal proximal tubular epithelial cells20 (link), and myoblast differentiation21 (link), we firstly established in vitro model of nicotine
(Sigma 36733) OA, by treatment of various doses of nicotine (100, 500, and 1,000
µM) to chondrocytes (passage 2) in the culture medium. After validating OA
properties in nicotine-treated chondrocytes, we further treated them with PDB
for 7 days.
replacement therapy. The osteoarthritic cartilage harvested from patients was
turned into pieces and digested with an enzymatic solution [8 mg/ml
hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), 8 mg/ml collagenase
(Sigma-Aldrich), and 2.5 mg/ml trypsin (Sigma-Aldrich)] for 6 h at 37°C. The
cellular suspension was centrifuged at 1,500 rpm for 5 min and resuspended into
Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, New York, NY, USA)
with 10% fetal bovine serum (FBS; Gibco BRL), and 1%
Penicillin-Streptomycin-Amphotericin B (PSA) (Biological Industries, Beit
Haemek, Israel) in a humidified atmosphere containing 5% CO2.
Thereafter, the primary OA chondrocytes were cultured, passaged, and maintained
in DMEM/F12 medium.
Based on previous reports showing various inhibitory activities of nicotine
against bone density19 (link), human renal proximal tubular epithelial cells20 (link), and myoblast differentiation21 (link), we firstly established in vitro model of nicotine
(Sigma 36733) OA, by treatment of various doses of nicotine (100, 500, and 1,000
µM) to chondrocytes (passage 2) in the culture medium. After validating OA
properties in nicotine-treated chondrocytes, we further treated them with PDB
for 7 days.
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