Dmem low glucose media

Colorectal cancer cell lines HT29 (ATCC® HTB-38), HT115 (ECACC 85061104), HCT116 (ATCC® CCL-247), and Caco2 (ATCC® HTB-37) as well as colonic epithelium CCD-841-CoN (ATCC® CRL-1790) cell lines were used in this study. Cell lines were maintained in DMEM low glucose media or MEM low glucose media (ThermoFisherScientific) supplemented with 10% (v/v) foetal bovine serum (ThermoFisherScientific). All cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

DMEM low glucose media, DMEM/F-12 media (1:1), Hams’ F-12 media, RPMI 1640 media, penicillin/streptomycin solution, phosphate buffered saline (PBS), Pierce RIPA buffer, and the Presto Blue cell viability reagent were purchased from ThermoFisher Scientific (Waltham, MA). Metformin and the media additives d-biotin, adenine hemisulfate, insulin solution, and apo-transferrin were purchased from Sigma Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Zapoglobin and Isoton II were purchased from Beckman Coulter Inc. (Fullerton, CA). Rabbit anti-mouse IgG secondary antibody was obtained from Zymed Laboratories, Inc. Both horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse antibodies were purchased from GE Healthcare Biosciences (Pittsburg, PA). All tissue culture plasticware and additional chemicals were purchased from ThermoFisher Scientific.

HepG2 cells were seeded into 6-well (30,000/cm²) plates for lipid accumulation assays. The cells were grown to confluence using Dulbec47co’s Modified Eagle Medium (DMEM) low glucose media (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), 1% Penicillin-Streptomycin-Neomycin Antibiotic Mixture (PSN, Thermo Fisher Scientific), and fetal bovine serum (FBS, Thermo Fisher Scientific). Then the cells were switched to an FBS-free media (DMEM and PSN only) for 48 h, followed by treatment of FBS-free media with 1% human plasma sample (PCOS Day 0 (PCOS-D0), PCOS Day 7 (PCOS-D7), CON Day 0 (CON-D0) or CON Day 7 (CON-D7)) added for an additional 48 h. Plasma samples added were from baseline draws on the respective days with a minimum of 18 h post previous ingestion of WPI supplementation. Plasma from 6 different participants from each group of the clinical part of this study were used for these experiments (one plasma source per well). Once HepG2 cells had been treated with human plasma for 48 h, lipid accumulation was measured using AdipoRed™ Assay Reagent (Lonza, Walkersville, MD, USA) per manufacturer’s instructions for a 6-well plate [47 ].

Under general anaesthesia, an aseptically created (2–3 cm) abdominal skin incision. Each dog’s autologous S/C fat (15–20 g) was extracted in sterile vessels. Chopped and thoroughly cleaned with phosphate-buffered saline to eliminate any debris or free blood, the obtained fat was then added to an equivalent amount of 0.1% collagenase type 1 (Sigma, Aldrich). The tissue was placed in an incubator that was constantly shaking at 37 °C for one hour. After assimilation, lipoaspirates were separated from collagenase by centrifuging for 10 min at 1200 rpm, and any enzyme that remained was washed away three more times. A hemocytometer was used to count the cells in the aspirates after the final round of centrifugation, and the trypan-blue-dye exclusion test was used to determine the viability of the cells [19 ]. When cells reached 70–80% confluency, they were trypsinized and divided to increase their number and were regarded as the first passage. When cells reached the third passage, they were harvested after trypsinization and characterized using flow cytometry markers CD90, CD 73, CD105, and CD 34. Cells were seeded in tissue culture flasks in DMEM low glucose media (Gibco) with 10% Fetal Bovine Serum, and the media was changed every three days [20 (link)]. Then, each case received an injection of the stem cell preparation (10 × 10⁶ cells in normal saline).

HeLa and female mouse embryonic fibroblast (MEF) cells were cultured in DMEM low glucose media (Gibco, 10567022) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (1 mg/mL) at 37 °C in a humidified incubator with 5% CO₂. For experiments, cells were seeded at a density of 10k cells per well in a 4-well cover-slipped bottom slide, 100k cells/well in a 6 well tissue culture plate, and 500k cells in a 10 cm plate. Cells were allowed to settle overnight and then an appropriate amount of MM-JH-1 was added for the desired final concentration from a 100 mM stock. Alternatively, the equivalent volume of DMSO was added as a negative control.