3T3-L1 cells were differentiated as described in the methods section for 9 days. The lipolysis assay was performed according to the manufacturer’s instructions (Abcam Lipolysis assay kit, ab185433). Briefly, after differentiation cells were washed two times with lipolysis assay buffer. Lipolysis was stimulated using 100 nM isoproterenol for 3 h. The amount of glycerol released was measured using colorimetric intensity.
Lipolysis assay kit
Manufactured by Abcam
The Lipolysis assay kit is a laboratory tool used to measure the rate of lipolysis, which is the breakdown of lipids into fatty acids and glycerol. The kit provides the necessary reagents and protocols to quantify the level of lipolysis in biological samples.
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10 protocols using lipolysis assay kit
1
Lipid Mobilization in 3T3-L1 Adipocytes
2
Evaluating Lipolysis in 3T3 L1 Cells
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Glycerol and free fatty acids released into the culture medium of differentiated 3T3 L1 cells were measured to evaluate lipolysis. In brief, differentiated 3T3 L1 cells with or without CREG1 knockdown were treated with BMS-345541 (5 μmol/L, Sigma), an NF-κB pathway inhibitor, or vehicle (dimethyl sulfoxide, DMSO, Sigma) for 48 hours prior to lipolysis assessment. The glycerol level was determined by Lipolysis Assay Kit (Abcam) and the free fatty acid level was measured by Free Fatty Acid Quantification Kit (Abcam) according to the manufacturer’s instruction.
3
Colorimetric Lipolysis Assay Protocol
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Lipolysis using colorimetric assay was performed using Abcam lipolysis assay kit following the manufacturer`s guidelines.
4
Plasma Lipid Regulation in 3T3-L1 Cells
5
Glycerol Quantification in Adipocytes
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Glycerol levels released into the culture medium were measured using a commercial kit (Lipolysis assay kit, ab185433, Abcam, Shanghai) according to the manufacturer’s instructions. The differentiated 3T3-L1 adipocytes were treated for 24 h with rutin. At room temperature, 50 μl of medium was incubated with 50 μl of reaction mixture for 30 min. The glycerol content was determined using a microplate reader by measuring absorbance at 570 nm. The measurements were made in triplicates, and the results are given in nmol/well.
6
Lipolysis Assay in 3T3-L1 Adipocytes with DREAM and Cytokine Exposure
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Lipolysis assay in 3T3-L1 adipocytes was performed after DREAM in all experiments. Experiments were conducted using the Abcam lipolysis assay kit (ab185433) as described by the manufacturer with adjustments for volumes suitable for 24-well plates. Briefly, cells were washed and kept in buffers supplied in the kit. For the different experiments, 10 nM of mFVIIa or 25 μM of PAR1 or PAR2 agonists was added for 30 min followed by incubation with 5 nM isoproterenol for 3 h. Glycerol released to the medium was measured by colorimetric absorbance read at 570 nm. For lipolysis estimations with long-term cytokine exposure, cells were DREAMed and allowed to settle for 24 h. Thereafter, cells were pre-stimulated with FVIIa (10 nM, 30 min) and subsequently kept for 48 h in growth medium supplemented with a mixture of TNF-alfa (10 ng/mL), IL-1 beta (5 ng/mL), and IFN-gamma (5 ng/mL). Lipolysis assay was thereafter performed as described above.
7
Quantifying Cellular Lipolysis Levels
8
Glycerol-based Lipolysis Quantification
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The lipolysis studies were conducted by measuring glycerol levels released into the cell culture medium, using commercial kit (Lipolysis assay kit, ab185433, Abcam, Shangai) following manufacturer's instructions [19] . The glycerol content was expressed as nmol/well.
9
Adipose-Derived Stem Cell Characterization
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Adipose derived mesenchymal stem cells were obtained from Himedia TM Cat.# CCK011. Complete medium and adipogeneic differentiation medium were also purchased from Himedia Cat.# AL521 and Cat.# AL537 respectively. Cytotoxicity kit was purchased from Promega TM Cat.# G1780 to check the cell viability. Free glycerol and Adiponectin were measured using Abcam's Lipolysis Assay kit (Cat. #ab185433) and Human Adiponectin ELISA kit (Cat.#ab99968). Other adipocytokines were quantified using the kits from Sigma Aldrich: Human Leptin Elisa kit (RAB0333), Human Interleukin-8(IL-8) kit (Cat. # RAB0319) and Human resistin ELISA kit (Cat. # RAb0419).
10
Glycerol Quantification in 3T3-L1 Cells
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Commercially available Abcam's Lipolysis Assay kit was used to measure glycerol released from 3T3-L1 cells. A volume of 0, 2, 4, 6, 8 and 10 µL of 1 mM glycerol standard was added into series of wells to generate 0, 2 ,4 ,6, 8 and 10 nmol/well of glycerol standards and the volume of each well was adjusted to 50 µL with Glycerol assay buffer followed by 50 µL of the reaction mixture. Incubation was done for 30 min and absorbance was read at 570 nm. Sample readings were applied to the standard curve to get nmol of glycerol amount in the sample wells.
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