Lipid extraction kit

Fresh fecal pellets were collected and weighed. Fecal lipids were extracted using a Lipid Extraction Kit (Cell Biolabs STA-162). Extracted lipids were air dried, resuspended in butanol, and quantified using a Lipid Quantification Kit for neutral lipids (Cell BiolabsSTA-617). Fecal carbohydrates were quantified using a Total Carbohydrate Assay Kit (Abcam, ab155891) according to the manufacture’s protocol.

The extraction of total lipids from cells exposed to compounds was performed using commercially available Lipid Extraction Kit (Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer protocol.
The content of unsaturated fatty acids in cells was performed using commercially available kit (Cell Biolabs Inc.) according to the manufacturer protocol.
The absorbance was measured at 550 nm using Bio-Tek microplate reader.

For testing glucose incorporation to lipids, preadipocytes isolated from 8-week-old C57BL/6J mice were treated with AdipoM for 3 days before being switched to insulin-only medium with or without PM or IWP2 for additional 8 days. Then, the uniformly labeled [U-¹⁴C₆] glucose (cat# ARC 0122G, American Radiolabeled Chemicals, St. Louis, MO, USA) was added at 0.2 μl/ml to the medium, with or without PM or IWP2, for three more days. Lipids were extracted with Lipid Extraction Kit (STA-612, Cell biolabs), and an equal volume of the extract was measured either with Lipid Quantification Kit (STA-613, Cell biolabs) or for radioactivity with a scintillation counter. The amount of ¹⁴C radioactivity was normalized to lipid quantity (cpm/μg). Glucose and lactate measurements were done as described (Esen et al., 2013 (link)). Briefly, for glucose consumption measurements, preadipocytes were incubated with AdipoM for 3 days and then switched to insulin-only medium with or without PM or IWP2 for three more days. Aliquots of the media and glucose standards were assayed with Glucose (HK) Assay Kit (Sigma catalog number GAHK20) and read at 340 OD using a plate reader (BioTek model SAMLFTA, Gen5 software). Lactate was measured with L-lactate assay kit from Eton biosciences (catalog number 1200011002). Each cell culture well was considered a biological replicate.

OvCa cells with the indicated treatments were collected and washed with PBS. Lipids were extracted from OvCa cells using a Lipid Extraction Kit (Cell Biolabs). The UFAs in cancer cells were detected at OD 540 nm, and the values were calculated according to the Lipid Quantification Kit protocol.

A total of 1 × 10⁶ hiPSC-CMs were treated with 100 μM palmitate acid for 48 h, followed by lipid extraction using the Lipid Extraction Kit (Cell Biolabs, STA-612) according to the manufacturer’s protocol. The isolated total lipids were dried in a vacuum concentrator at 4 °C and resuspended in 700 μl of TRIzol. RNA was then extracted by using the RNeasy mini kit (Qiagen, 74106).