The phospholipase assay was carried out as described previously [45 ]. Membrane PLs were extracted from photoheterotrophically grown R. sphaeroides [46 (link)] and used as substrates for the assay. The reaction mixture contained an aliquot (35 µg protein) of either the cytoplasmic or periplasmic fraction (“Preparation of cytoplasmic and periplasmic fractions ” section) and 400 µg of PLs in 1 mL of 20 mM sodium borate buffer (pH 9.0) containing 1 mM CaCl2. The reaction was performed at 30 °C for 15 min, and terminated by heating at 100 °C for 5 min. The reaction mixture was then centrifuged at 12,000g and 4 °C for 10 min, and the supernatant (50 µL) was examined for FFA using the FFA Quantification Colorimetric/Fluorometric Kit (BioVision, Milpitas, CA, USA). The reaction was performed with no cell fraction aliquot as a control, and a standard curve was prepared with varying concentrations of palmitate. One enzyme unit was defined as 1 µmol of FFAs released per min from PLs.
Ffa quantification colorimetric fluorometric kit
Manufactured by Abcam
Sourced in United States
The FFA Quantification Colorimetric/Fluorometric Kit is a laboratory tool used to quantify the levels of free fatty acids (FFAs) in various biological samples. The kit provides a quick and reliable method for measuring FFAs through either a colorimetric or fluorometric assay.
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4 protocols using ffa quantification colorimetric fluorometric kit
1
Phospholipase Assay for Rhodospirillum sphaeroides
2
Cellular Free Fatty Acid Quantification
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Cellular FFA was measured by using an FFA quantification colorimetric/fluorometric kit (cat no. K612, Biovision, Mountain View, CA, USA) according to manufacturer's indication. Same numbers of UCB-hMSC samples were collected and incubated with acetyl-CoA synthetase reagent, enhancer solution, and enzyme mixture as provided in the kit. Lipid samples were incubated at 37 °C for 30 min. Cellular FFA levels were measured by using a microplate reader at 550 nm (Bio-Rad).
3
Quantification of Fatty Acids in SACC Cells
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The contents of FFAs were determined using an FFA quantification colorimetric/fluorometric kit (BioVision, Waltham, MA, USA). The palmitic acid standard liquids in the kit were diluted to 0, 0.2, 0.4, 0.6, 0.8, or 1.0 nmol per well. Subsequently, 10 mg SACC tissue samples or 106 SACC‐83 cells were homogenized with 200 µL chloroform‐Triton X‐100. The extracts were centrifuged and dried for 10 min. The dried lipids were dissolved in a 200 µL fatty acid assay buffer. The absorbance at a wavelength of 570 nm was determined for colorimetric assay, and Ex/Em = 535/590 nm was applied for fluorescence measurements in a microplate reader.
4
Adipose Tissue Biomarker Quantification
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The subcutaneous WAT (scWAT) was homogenized in tissue protein extraction reagent. Total protein was extracted and the concentration was measured using BCA protein quantification kit. The content of cytokines and norepinephrine in local scWAT were determined by ELISA kits. IL-4 was measured by mouse IL-4 ELISA kit (#431107, BioLegend). IL-5 and IL-33 were measured by Platinum ELISA (#BMS610, #BMS6025, eBioscience). Norepinephrine was measured by 2-CAT (A-N) Research ELISA (#BA-E5400, LDN). Circulating Leptin and Insulin were measured by Crystalchem INC and circulating NEFA was measured by FFA Quantification Colorimetric/Fluorometric Kit (#K612, Biovision). Triglycerides (GPO-PAP method), total cholesterol (CHOD-PAP method), high density lipoprotein cholesterol (direct method) and low-density lipoprotein cholesterol (direct method) in circulating were analyzed on the auto clinical chemistry analyzer platform (ZY-330, KHB). Blank Control and quality Controls were added in every measurement. All the processes were administrated according to the manufacturer's instructions.
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