WT or CB1R‐/‐‐HK‐2 cells (human immortalized RPTCs) were maintained in a low glucose DMEM (01‐050‐1A; Biological Industries, Beit Haemek, Israel) supplemented with 5% FCS, 100 mM glutamine, 100 mM Na‐pyruvate and Pen/Strep (Thermo Fisher Scientific, UK). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air. To test the effect of CB1R activation, cells were seeded in 6‐well plates (25 × 104 cells/well) for 24 hours. Then, growth medium was replaced with serum‐free medium (SFM) for an additional 12 hours. On the morning of the experiment, the medium was replaced with fresh SFM containing either vehicle (EtOH), 5 μM AEA, 5 μM ACEA or 250 nM JZL195 for 6 hours. To mimic fatty conditions, cells were overloaded with a mixture of fatty acids containing 0.1 mM sodium Oleate (O7501; Sigma Aldrich, St. Louis, Missouri) and sodium Palmitate (P9767; Sigma Aldrich) in a ratio of 2:1 (O:P, respectively), dissolved in 11% free‐fatty acid BSA solution (A7030; Sigma Aldrich). At indicated time points, cells were harvested for further analyses as described below.
O7501
Manufactured by Merck Group
Sourced in Germany, China
The O7501 is a precision laboratory equipment designed for conducting various scientific experiments and analyses. It features advanced technology and high-quality components to ensure accurate and reliable performance. The core function of the O7501 is to provide a controlled and stable environment for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
P9767, by Merck Group (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Low glucose dmem, by Sartorius (1 mentions)
A7030, by Merck Group (1 mentions)
P0500, by Merck Group (1 mentions)
M3128, by Merck Group (1 mentions)
S2770, by Merck Group (1 mentions)
P9417, by Merck Group (1 mentions)
D4902, by Merck Group (1 mentions)
E1013, by Applygen (1 mentions)
Fetal bovine serum (fbs), by Cellmax (1 mentions)
C0222, by Beyotime (1 mentions)
Sa211, by Cellmax (1 mentions)
St025, by Beyotime (1 mentions)
C11995500bt, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Beyotime (1 mentions)
Streptomycin, by Beyotime (1 mentions)
C11995500cp, by Thermo Fisher Scientific (1 mentions)
6 protocols using o7501
1
CB1R Activation in Human RPTCs
2
Modulation of Type I Interferon Response by Fatty Acids
3
Glucocorticoid Regulation of Hepatic Lipid Metabolism
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HepG2 cells, obtained from Keygen Biotech (Nanjing, China, ATCC HB-8065), were maintained in DMEM medium containing 10% FBS and 1% P/S at 37 °C with 5% CO2 (Thermo Scientific, CO2 incubator) in 75 cm2 flasks. Cells were plated in 6-well plates at 2*105 cells per well. The following day, confluent cells were starved for 6 h without FBS. Then, the cells were treated with 2.0 ml of fresh supplemented culture medium containing dexamethasone (active GC, D4902, Sigma), mifepristone (glucocorticoid receptor (GR) antagonist, Mi, M8046, Sigma), both GC and Mi, or vehicle (culture medium) for 48 h, followed by exposure (or not) to sodium oleate (OA, O7501, Sigma), which is rich in fatty acids, for 24 h. To evaluate the possible effects of GC on gene expression related to lipid metabolism, HepG2 cells were incubated with GC at different concentrations (0, 50, 100, 125, 250, 500 and 1000 nM; n = 3 for each concentration) and time (24, 36 and 48 h) and to ascertain the maximal response. The effects of GC (125 nM) combined with Mi at different concentrations (0, 0.1, 1, 5, 10 μM) were then used (n = 3) to evaluate the individual and combined effects on the hepatic lipid homeostasis. The TG content in the cells was determined using commercial kits (E1013, Applygen, Beijing, China).
4
Inducing Hepatic Steatosis in HepG2 Cells
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HepG2 hepatocyte cells were cultured at 37°C, 5% CO2, and constant pH (7.2-7.4) in Dulbecco’s Modified Eagle Medium (DMEM; C11995500BT, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; SA211.02, Cellmax, Australia), 100 U/mL penicillin, and 100 μg/mL streptomycin (C0222, Beyotime, China). OA (O7501, Sigma, Germany) was conjugated to 10% (v/v) bovine serum albumin (BSA) (ST025, Beyotime, China). To induce hepatic steatosis, HepG2 cells were exposed to 0.5 mM OA for 24 h to induce lipid accumulation (62 (link)–64 (link)). Cells were then washed, cultured in fresh complete medium, and treated with different drugs for further analysis.
5
Oleic Acid Treatment of Hepatocyte Cell Lines
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Hepatocyte cell lines Hepa 1–6 and Hep 3B and 293T cells were cultured in Dulbecco's modified Eagle medium (C11995500CP; Gibco, Beijing, China) supplemented with 10% fetal bovine serum (10099-141; Gibco) as well as 100 U/mL penicillin and streptomycin in 5% CO2 at 37°C. The culture medium was changed every 2 days unless otherwise stated.
For OA (O7501; Sigma-Aldrich, Shanghai, China) treatment, 10 mmol/L OA stock solution was added to the culture medium to a final concentration of 0.1 or 0.2 mmol/L, and then the cells were treated for either 6, 12, or 24 hours according to the specific experimental purpose.
For OA (O7501; Sigma-Aldrich, Shanghai, China) treatment, 10 mmol/L OA stock solution was added to the culture medium to a final concentration of 0.1 or 0.2 mmol/L, and then the cells were treated for either 6, 12, or 24 hours according to the specific experimental purpose.
6
Fatty Acids and Beta-Cell Function
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Sodium salts solutions of nonesterified palmitic (Sigma, code P9767) and oleic (Sigma, code O7501) acids were prepared as previously reported [34 (link)]. Briefly, 1 mL of 100 mM palmitic or oleic solutions (27.8 mg of palmitate and 30.4 mg oleate) was dissolved in NaOH 0.1 M and warmed up to 60°–70°C and gently shaken. Then, the solutions were diluted twenty times in DMEM medium with 10% fatty acid-free Bovine Serum Albumin (BSA). Diluted NEFAs solutions were filtered (0.45 μm), aliquoted in amber tubes, and frozen at −20°C. Experiments were then conducted by diluting 10x with fresh and sterile DMEM medium (10% fetal bovine serum, FBS) to reach the final concentration of 0.5 mM of NEFAs salts and 1% BSA.
MIN6 β-cells were incubated with palmitate or oleate (0.5 mM, 1% BSA) in DMEM medium (10% FBS) for 24 hours, and then glucose-stimulated insulin secretion was assessed. In parallel, the viability of MIN6 β-cells after 24 h of exposure to fatty acids was measured by Trypan Blue exclusion assay and the percentage of viability was quantified by an automated cell counter (Luna, Logos Biosystems).
MIN6 β-cells were incubated with palmitate or oleate (0.5 mM, 1% BSA) in DMEM medium (10% FBS) for 24 hours, and then glucose-stimulated insulin secretion was assessed. In parallel, the viability of MIN6 β-cells after 24 h of exposure to fatty acids was measured by Trypan Blue exclusion assay and the percentage of viability was quantified by an automated cell counter (Luna, Logos Biosystems).
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