Fatty acid free bsa

The IVF procedure was that mature oocytes were washed twice or three times in Brackett and Oliphant (BO) [78 (link)] fertilization medium which contained 4 mg/mL fatty acid-free BSA (Sigma-Aldrich, St. Louis, MO, USA) and 10 μg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA). Oocytes were transferred into a 90 μL drop of BO fertilization medium at a density of 20–25 COCs per group. Frozen semen was thawed in a water bath at 37 °C for 30 s, rinsed twice in 7 mL BO medium containing 4 mg/mL fatty acid-free BSA and 10 mmol/L caffeine (Sigma-Aldrich, St. Louis, MO, USA) by centrifugation at 1500 rpm for 5 min, and finally resuspended at a concentration of 1 × 10⁶ spermatozoa per mL. For IVF, a 10 μL aliquot of the sperm suspension was added to each fertilization drop and the mixture was incubated for 16–18 h at 38.5 °C in humidified air containing 5% CO₂.

Palmitate was first dissolved in DMSO (Millipore, Cat # 41639), and subsequently this solution was dissolved at 45 °C in DMEM media (no glucose) containing 6.7% fatty acid-free BSA (EMD Millipore, Cat # 126609) to make a 4 mM (10×) stock. For control BSA conditions, a 10× stock of DMEM media containing 5% BSA and 1% DMSO was used. For the treatment conditions, the 10× stocks were added to DMEM media containing 5% FBS, 50 U/mL penicillin, and 50 g/mL streptomycin. The pH of the treatment media was then adjusted to 7.4, and the adjusted media was sterile filtered using a 0.45 µm syringe filter (Millipore, Cat # SLHV004SL) before treating the HepG2 cells for 16 h.

PPARγ ligand inhibition constants (K_i) were measured using a protocol adapted from LanthaScreen TR-FRET PPARγ competitive binding assay (Invitrogen, catalog number PV4894). Assay was performed by plating a mixture of 8 nM 6xHis-PPARγ-LBD, 2.5 nM LanthaScreen Elite Tb-anti-His antibody, 5 nM LanthaScreen Fluormone Pan-PPAR Green (Invitrogen, catalog number PV4896), and 12-point serial dilutions of PPARγ ligands from 50 μM to 140 fM. This mixture was added to wells of low-volume 384-well black plates (Grenier Bio-one) to a final volume of 16 μL. All dilutions were made in 25 mM MOPS (pH 7.4), 25 mM KCl, 1 mM EDTA, 0.01% fatty-acid free BSA (EMD Millipore), 0.01% Tween, and 5 mM TCEP. Assay titrations were performed in duplicate. Plates were incubated in the dark for 2 h at room temperature before being read on a Synergy H1 microplate reader (BioTek). TR-FRET was measured by excitation at 330 nm/80 nm and emission at 495 nm/10 nm for terbium and 520 nm/25 nm for Fluormone. Change in TR-FRET was calculated by 520 nm/495 nm ratio. Nonlinear curve fitting was performed using Prism 7.0b (Graphpad Software Inc.) as described above for the TR-FRET data, including manual exclusion of highest two concentrations for nTZDpa. Thirty of the 1224 total data points for all three proteins (wt, PPARγ^K502C-BTFA, and PPARγ^C313A,K502C-BTFA) were automatically excluded by Prism in the fits.

U87-MG cells plated at 6 × 10⁴ cell/cm² were grown on a glass coverslip and maintained for 24 h in DMEM with 10%FBS. After 48 h of treatments with or without 8 µM of TubA plus 10 µM of cyclo, cells were incubated at 4 °C for 30 min with 5 μM of N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoyl) Sphingosyl Phosphocholine, (BODIPY-C5-SM; Thermo Fisher Scientific Inc. Waltham, MA, USA) as a complex with fatty acid-free BSA (1:1, mol:mol) in Krebs Ringer buffer at a pH of 7.4. At the end of loading, after two washes with DMEM with 10% FBS and 0.34 mg/mL of fatty acid-free BSA (Merck KGaA Darmstadt, Germany), cells were incubated for different durations at 37 °C. Cells were then washed three times with PBS and fixed with 0.5% glutaraldehyde solution for 10 min at 4 °C. After washes in PBS at a pH of 7.4 at 4 °C, the specimens were immediately observed and analyzed with a fluorescence microscope (Olympus BX-50).