Free fatty acid assay kit

Pregnant mice were
fasted for 16 h. The fasting blood glucose levels were measured using
an Accu-Check glucometer (Roche, US). A Free Fatty Acid Assay Kit
(Abcam) and a Triglyceride Colorimetric Assay Kit (Cayman Chemical)
were used to measure serum fatty acids and liver triglycerides, respectively.
For ATP measurement, liver samples were homogenized in 1× passive
lysis buffer (Promega), followed by the measurement according to the
manufacturer’s instructions (ATP Determination Kit, Invitrogen).
Sample protein concentrations were measured using a DC Protein Assay
Kit II (Bio-Rad). Luminescence was determined by using a PerkinElmer
EnSight Multimode Plate Reader.

For the detection of FFA in samples, the Free Fatty Acid Assay Kit was used (Cat. ab65341, Abcam, Waltham, MA). For lung samples, FFAs were extracted using Lipid Extraction Kit (Cat. Ab211044, Abcam, Waltham, MA) from equal weight of lung tissue samples (40 mg). Serum samples were directly assayed. The FFA Assay Kit converts fatty acids into their CoA-derivatives which are then oxidized with the concomitant generation of color or fluoresence. Fifty (50) μl of samples (dissolved in supplied assay buffer if needed) were added to each well in a 96-well plate. The acyl-CoA synthesis was then performed by adding 2 μl of ACS reagent into the wells, mixed, and incubated at 37 °C for 30 min. A reaction mix (44 μl assay buffer, 2 μl fatty acid probe, 2 μl enzyme mix, and 2 μl enhancer was added to each well and incubated at 37 °C for 30 min in the dark. After incubation the absorbance was measured at 570 nm (SpectraMax 190 Microplate Reader, Molecular Devices). A standard curve was generated using palmitic acid and the concentration of fatty acid was determined using formula, [FFA] = Fa/Sv (nmol/μl or mM) where Fa is the fatty acid amount in the well obtained from the standard curve and Sv is the sample volume (μl) added to the sample well. For lung samples, the amount of FFA was expressed per weight (g) of lung.

Serum measurements for the following tests were taken at the beginning of treatment (baseline) and weekly until the end of treatment and performed according to local standard procedures: standard of care clinical assays for PSA, testosterone, lipid profiling, and serum biochemistry. Serum FFA levels were measured using the Free Fatty Acid Assay Kit (Abcam, Cambridge, UK) as per the manufacturer's instructions. For clinical reasons, serum samples for biochemical analysis were not available for Patient 015, so FFA levels were not measured for this patient.

Both WT and TG mice were fasted for 6 h before sacrifice, and dissected quadriceps muscle and blood were obtained. TAG, triglycerides and FFAs were assayed using a Triglyceride Assay Kit (ABCAM) and a Free Fatty Acid Assay Kit (ABCAM). The levels of TAG, triglycerides, and FFAs were measured by the fluorescent intensities at an absorbance ratio of 535/590 nm wavelength. Finally, the levels of TAG, triglycerides, and FFAs were calculated based on typical standard curves.

Lipolysis was assessed as previously described^{4 (link)}. Inguinal and epididymal fat depots were surgically removed from 2-month-old control and Ai-α4KO at 6 days after the last injection of tamoxifen. After washing with cold PBS, a fragment of 20–30 mg was further cut into five or six pieces and incubated for 2 h at 37 °C in 200 μl of DMEM containing 2% fatty acid-free BSA (Sigma-Aldrich) in the presence or absence of 10 mM isoproterenol (Sigma-Aldrich). Fatty acids released into the medium were quantified using a free fatty acid assay kit (Abcam, cat. no. ab65341) and normalized to the weight of each fat pad.