Diisononyl phthalate

The eleven phthalates and the two non-phthalates studied plasticizers are listed in Table 1. Benzyl butyl phthalate (BBP; ≥98%), dibutyl phthalate (DBP; ≥99%), di-cyclohexyl phthalate (DCHP; ≥99%), di(2-ethylhexyl) phthalate (DEHP; ≥99.5%), di-(2-ethylhexyl) terephthalate (DEHT; ≥97%), diethyl phthalate (DEP; ≥99.5%), diethyl phthalate-3,4,5,6-d₄ (DEP-d₄, IS; ≥98%), diisobutyl phthalate (DIBP; ≥99%), diisononyl phthalate (DINP; ≥99%), dimethyl phthalate (DMP; ≥99%), di-n-octyl phthalate (DNOP; ≥98%), diphenyl phthalate (DphP; ≥99%), dipropyl phthalate (DPP; ≥98%), and tri-octyl trimellitate (TOTM; ≥99%) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). HPLC-grade solvents were used. Acetone, dichloromethane, and acetonitrile were purchased from VWR (Fontenay-sous-Bois, France). Ultra-pure water was supplied by a Milli-Q purification system from Millipore (St Quentin en Yvelines, France).

Adiponectin and resistin secretion were measured in mouse primary adipocytes isolated from inguinal white adipose tissue (30-min incubations at 32 °C). Primary adipocytes were isolated from 8-week-old mice inguinal white adipose tissue. Tissue samples were minced and digested using collagenase type II (1 mg/mL; 45–60 min; 37 °C) and poured through a 100-μm nylon mesh. Floating adipocytes were washed with Krebs–Ringer glucose buffer (1% BSA) and immediately used for experiments. Primary adipocytes were diluted to 25% volume/volume. Secretion was measured in an EC (see [Ca²⁺]_i imaging) containing test substances as indicated. Primary adipocytes cells were separated from media by centrifugation in diisononyl phthalate (Sigma–Aldrich) followed by snap freezing in dry ice. Tubes were cut through the oil layer at two points. Cells were lysed in PBS containing SDS (2%) and protease inhibitor (1 tablet/10 mL; PhosSTOP, Sigma Aldrich). EC aliquots and cell homogenates were stored at −80 °C. Secreted adiponectin or resistin (measured with mouse ELISA DuoSets; R&D Systems) was expressed in relation to total protein content (BCA protein assay).