Free fatty acid uptake assay kit

Manufactured by Abcam

Sourced in United States, United Kingdom

The Free Fatty Acid Uptake Assay kit is a laboratory tool designed to quantify the uptake of free fatty acids by cells. The kit provides a fluorescence-based method to measure the kinetics and extent of fatty acid internalization, allowing researchers to study cellular lipid metabolism.

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Lab products found in correlation

Envision 2104, by PerkinElmer (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Wallac 1420 victor2 microplate reader, by PerkinElmer (1 mentions) Cellular ros assay kit, by Abcam (1 mentions) Varioskan lux, by Thermo Fisher Scientific (1 mentions) Triglyceride quantification colorimetric fluorometric kit, by Abcam (1 mentions) Phosphatidylcholine assay kit, by Abcam (1 mentions) Spectramax microplate reader, by Molecular Devices (1 mentions) Glutamine assay kit, by Abcam (1 mentions) Advanced dmem, by Thermo Fisher Scientific (1 mentions) Glucose colorimetric fluorometric assay kit, by Abcam (1 mentions)

9 protocols using free fatty acid uptake assay kit

Fatty Acid Uptake and Visualization

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Fatty acid uptake was measured using a Free Fatty Acid Uptake Assay kit (ab176768, Abcam, USA), following the manufacturers’ protocol. Briefly, cells were seeded at a density of 50,000 cells per well in a 96-well plate 1 day before measurement, and these cells were deprived of serum for 1 hour. Next, cells were incubated with TF2-C12 fatty acid at room temperature, and fluorescence (excitation: 485 nm, emission: 515 nm) was measured using a Wallac 1420 Victor² Microplate Reader (Perkin Elmer, USA).
For the fluorescent FA pulse-chase assay, cells were deprived of serum for 1 hour and pulsed with Red C12 overnight in order to allow the Red C12 to accumulate in lipid droplets. After washing with PBS, cells were measured with confocal microscopy.

Su P., Wang Q., Bi E., Ma X., Liu L., Yang M., Qian J, & Yi Q. (2020). Enhanced lipid accumulation and metabolism are required for the differentiation and activation of tumor-associated macrophages. Cancer research, 80(7), 1438-1450.

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Fatty Acid Uptake in CRC Cells

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Fatty acid uptake was evaluated with a Free Fatty Acid Uptake Assay Kit (Abcam, ab176768) according to the manufacturer’s protocol. Briefly, CRC cells were plated in a 96-well plate at a density of 5 × 10⁴ cells/well and cultured for 10 h. Then, cells were preincubated in serum-free media for 1 h and subsequently incubated with fluorescent fatty acid. After 50 min, the fluorescence intensity was detected by a microplate fluorescence reader at 485/515 nm.

Yang Y., He J., Zhang B., Zhang Z., Jia G., Liu S., Wu T., He X, & Wang N. (2021). SLC25A1 promotes tumor growth and survival by reprogramming energy metabolism in colorectal cancer. Cell Death & Disease, 12(12), 1108.

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Free Fatty Acid Uptake Assay

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Free fatty acid uptake and transportation activities were assessed using the Free Fatty Acid Uptake Assay Kit (#ab176768, Abcam) following the manufacturer’s instructions. Briefly, L02 cells were transfected with pcDNA3.1(+) or GSTA1 plasmids and incubated for 48 h. The transfected L02 cells were then seeded into a 96-well plate, allowed to adhere for 6 h, and treated with or without bicyclol for 30 min. After washing with phosphate-buffered saline, the cells were incubated in serum-free medium for 1 h and then treated with a fluorescent fatty acid mixture for 30 min. Fluorescence intensity was measured using a microplate fluorescence reader with bottom-read mode at 515 nm after excitation at 485 nm. The fluorescence signal of the control group was used as a baseline for relative quantification.

Jiang J., Li H., Tang M., Lei L., Li H.Y., Dong B., Li J.R., Wang X.K., Sun H., Li J.Y., Xu J.C., Gong Y., Jiang J.D, & Peng Z.G. (2024). Upregulation of Hepatic Glutathione S-Transferase Alpha 1 Ameliorates Metabolic Dysfunction-Associated Steatosis by Degrading Fatty Acid Binding Protein 1. International Journal of Molecular Sciences, 25(10), 5086.

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Fatty Acid Uptake Assay in hiPSC-CMs

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4 days prior to the assay, 2x10⁴ hiPSC-CMs were seeded on a Matrigel coated 384-well plate. The fatty acid was evaluated with the Free Fatty Acid Uptake Assay Kit (Abcam, ab176768) as recommended by manufacturer’s protocol. Cells were washed 3x with serum-free media and preincubated in serum-free media for 2 hours. A baseline measurement was preformed prior to the addition of the fluorescent fatty acid mixture, and every 10 mins after the addition by using a microplate fluorescence reader at 485/528 nm(PerkinElmer, EnVision 2104).

M. Feyen D.A., McKeithan W.L., N. Bruyneel A.A., Spiering S., Hörmann L., Ulmer B., Zhang H., Briganti F., Schweizer M., Hegyi B., Liao Z., Pölönen R.P., Ginsburg K.S., Lam C.K., Serrano R., Wahlquist C., Kreymerman A., Vu M., Amatya P.L., Behrens C.S., Ranjbarvaziri S., C. Maas R.G., Greenhaw M., Bernstein D., Wu J.C., Bers D.M., Eschenhagen T., Metallo C.M, & Mercola M. (2020). Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes. Cell reports, 32(3), 107925.

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Fatty Acid Uptake and ROS Assay

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To perform the fatty acid uptake assay, primary adipose cells were isolated from epididymal adipose tissue as described previously (21 (link)). After seeding in 96-well plates, cells were incubated with serum-free DMEM for 1 h. Fatty acid uptake was assessed using a Free Fatty Acid Uptake Assay kit (catalog no.: ab176768; Abcam, Cambridge, UK) according to the manufacturer’s instructions. Fluorescence signals were measured using a fluorescence microplate reader (Varioskan LUX; Thermo Fisher Scientific) at 485/515 nm.
Cellular reactive oxygen species (ROS) level was measured using a DCFDA (2',7'-dichlorofluorescein diacetate)/H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) Cellular ROS Assay kit (catalog no.: ab113851; Abcam) as per the manufacturer’s instructions. If required, cells were preincubated with hydrogen peroxide (H₂O₂) or oleic acid for 24 h before the assay. Fluorescence signals were measured at 485/515 nm.

Yoo A., Joo Y., Cheon Y., Lee S.J, & Lee S. (2022). Neuronal growth regulator 1 promotes adipocyte lipid trafficking via interaction with CD36. Journal of Lipid Research, 63(6), 100221.

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Fluorometric FFA Uptake Assay

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FFA uptake rate was measured using the Free Fatty Acid Uptake Assay kit (Abcam). In brief, 3T3-L1 preadipocytes were serum-starved for 1 h, and then incubated with FA dye-loading solution for 10 min. Fluorescence signals were measured using a microplate reader with FITC filter (PerkinElmer).

Xu D., Li Y., Wu L., Li Y., Zhao D., Yu J., Huang T., Ferguson C., Parton R.G., Yang H, & Li P. (2018). Rab18 promotes lipid droplet (LD) growth by tethering the ER to LDs through SNARE and NRZ interactions. The Journal of Cell Biology, 217(3), 975-995.

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Fatty Acid Uptake Assay in hiPSC-CMs

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Lipid Characterization of iHLCs

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Cell-based fluorometric assays were performed using the Free Fatty Acid Uptake Assay kit (#ab176768, Abcam). Briefly, iHLCs were grown in 96 well plate and incubate 1 h in serum-free media. Fatty acid dye-loading solution was added for 4 h and the fluorescence signal measured with a SpectraMax fluorescence microplate reader at Ex/Em = 485/515 nm.
Triglyceride content of iHLCs was measured using the Triglyceride Quantification Colorimetric/Fluorometric kit (# K622-100, Biovision). iHLCs(10⁶ cells) were suspended and lysed in 5% NP-40/ddH2O solution. The output was measured at OD 570 nm for colorimetric assay after mixing and incubating with assay reagents provided with the kit.
Phosphatidylcholine in iHLC cell lysates was measured using the Phosphatidylcholine Assay kit (#ab83377, Abcam) and a SpectraMax Microplate reader (Molecular Devices). Briefly, iHLCs were grown in 96 well plates, washed in PBS and suspended in assay buffer provided with the kit. Both standards and samples were incubated with kit components as directed and the measurement was performed at OD570 (OD is optical density).

Mukhopadhyay B., Marietta C., Shen P.H., Oiseni A., Mirshahi F., Mazzu M., Hodgkinson C., Winkler E., Yuan Q., Miranda D., Kunos G., Sanyal A.J, & Goldman D. (2024). A patient-based iPSC-derived hepatocyte model of alcohol-associated cirrhosis reveals bioenergetic insights into disease pathogenesis. Nature Communications, 15, 2869.

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Quantifying Metabolic Profiles of Organoid Cultures

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There are 5×10³ organoids were cultured in 96-well plates containing advanced DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 2 mM glutamine and then transferred to a CO₂ incubator set at 37 °C and 5% CO₂ for 24 hours. Spent media were collected to measure remaining glucose, fatty acid and glutamine by using a glucose colorimetric/fluorometric assay kit (Abcam), free fatty acid uptake assay kit (Abcam) and glutamine assay kit (Abcam) respectively following the manufacturer's instruction.

Wang R., Xiang W., Xu Y., Han L., Li Q., Dai W, & Cai G. (2020). Enhanced glutamine utilization mediated by SLC1A5 and GPT2 is an essential metabolic feature of colorectal signet ring cell carcinoma with therapeutic potential. Annals of Translational Medicine, 8(6), 302.

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