We used a modified version of a well-established lipolysis assay [36] (link) to evaluate NEFA, glycerol, and leptin release from adipose organ explants. Briefly, eWAT, iWAT, and BAT from Adipoq-GsD, Adipoq-GiD and respective littermate controls were surgically removed and cut in ∼20 mg pieces. The pieces were put in a 96-well plate containing 200 μl of prewarmed (37 °C) DMEM no phenol red (Gibco A1443001). The pieces were then incubated at 37 °C (5% CO2, and 95% humidified atmosphere) for one hour (baseline) in DMEM containing 2% fatty acid (FA)-free bovine serum albumin (BSA, Sigma, A8806) and 0.1% glucose (Sigma 49163). To avoid re-esterification of FA and glycerol, each well also contained 5 μM of the acyl-CoA synthetases inhibitor triacsin C (Tocris 2472). At the end of the hour, the pieces were transferred to a new 96-well plate containing fresh medium with 1 μM CNO (C0832, Sigma) and incubated for another hour (stimulated). The incubation media from baseline and stimulated conditions were then used to evaluate NEFA release by colorimetry (FUJIFILM Wako Diagnostics-NEFA Reagent, 999-34691, 995-34791, 991-34891, 993-35191), glycerol release by colorimetry (F6428, Sigma), and leptin release by ELISA (Mouse/Rat Leptin ELISA, ALPCO, 22-LEPMS-E01). NEFA, glycerol, and leptin release were then calculated based as a percentage compared to baseline.
Dmem no phenol red
Manufactured by Thermo Fisher Scientific
Sourced in United States
DMEM (no phenol red) is a cell culture medium that is formulated without the addition of phenol red indicator. It is designed to support the growth and maintenance of a variety of cell lines in vitro. The core function of this product is to provide a balanced nutritional environment for cell culture applications that do not require the presence of the pH indicator phenol red.
Automatically generated - may contain errors
Lab products found in correlation
A8806, by Merck Group (1 mentions)
F6428, by Merck Group (1 mentions)
C0832, by Merck Group (1 mentions)
Triacsin c, by Bio-Techne (1 mentions)
Mouse rat leptin elisa, by ALPCO (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Glycerol assay kit, by Nanjing Jiancheng (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Dmem f12 no phenol red, by Thermo Fisher Scientific (1 mentions)
5 protocols using dmem no phenol red
1
Adipose Tissue Lipolysis Assay
2
Visualizing Bile Canaliculi Formation
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To visualize bile canaliculi formation, cells were rinsed twice with 1X DPBS (–Ca++/–Mg++) (Thermo Fisher). Fresh medium plus 5 µM 5- (and-6)-Carboxy-2’, 7’-Dichlorofluorescein Diacetate (CDFDA) (Thermo Fisher) were added to the cells and incubated for 20 min at 37 °C. Cells were then washed twice with 1X DPBS (–Ca++/–Mg++), and DMEM (no phenol red) (Gibco) was added to the cells. Images were taken with an EVOS FL cell imaging system (Thermo Fisher) using a 10X or 20X objective.
3
HCT116 Cell Growth and Viability Assay
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HCT116 cells were seeded into six-well plates and were counted every 24 h to establish cell growth curves. HCT116 cells were seeded into 6 cm dishes. After culturing for 7 days, cells were washed with PBS three times, fixed with 4% paraformaldehyde, and stained with 0.2% crystal violet. Pictures were shot to show colony formation. HCT116 cells were seeded into 96-well plates and cultured for 48 h. 10 μl CCK-8 was added into 100 μl DMEM (no phenol red, Gibco, USA) in each well. The plate was incubated for 1 h in the incubator (37 °C, 5% CO2). The absorbance at 450 nm (A450) was finally measured using microplate reader.
4
Irisin-Induced Glycerol Secretion
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The treated cells were washed once with PBS and were then incubated with DMEM (no phenol red) (Gibco, USA) with or without GST-irisin for 4 h. Samples of the media were collected and assayed for glycerol levels using a glycerol assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
5
Cell Culture Conditions for Cancer Models
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Human ER + MCF7 breast cancer cells (HTB-22), were grown in Dulbecco's modified eagle medium (DMEM) no phenol red (Gibco, Cat# 21063029), 10 % fetal bovine serum (FBS) (VWR, Cat# 1300-500), 5 mL penicillin-streptomycin (Gibco, Cat# 15070063). Rat-derived fibroblast lines were grown in DMEM/F12 no phenol red (Gibco, Cat# 15070063), 10 % FBS, and 5 mL gentamycin (Gibco, Cat# 15750060). Rat adenocarcinoma-derived tumor cell lines were grown in MEC media, which is DMEM/F12 no phenol red (Gibco, Cat# 21041-025), 10 % horse serum (Gibco, Cat# 16050-122), 20 ng/mL EGF (Sigma, Cat# E9644), 0.5ug/mL hydrocortisone (Sigma, Cat# H088), 100 ng/mL cholera toxin (Sigma, Cat# C8052), and 10ug/mL insulin (Sigma, Cat# I6634). All cells were grown at 37 °C in a 5 % CO2 incubator. Mycoplasma testing (ATCC, Cat# 30-1012-K) every 6 months confirmed cells were not infected.
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