Rat insulin elisa kits

Insulin content was measured as described before [22 (link)]. Briefly, INS-1E cells were seeded on cell culture 24-well plates at a density of 5 × 10⁵ cells per well and cultured overnight. The cells were washed twice with PBS and incubated further with RPMI-1640 medium containing different concentrations of either TNF-α (0, 2, and 20 ng/ml; R&D Systems, Oxon, UK), or leptin (0, 0.5, and 10 nM; R&D Systems, Oxon, UK), or combination of TNF-α and leptin. After 48 h of incubation, the cells were washed twice with Krebs-Ringer bicarbonate buffer (KRBB, 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 5 mM NaHCO₃, 0.1% BSA, and 10 mM HEPES [pH 7.4]) and starved for 2 hours in KRBB, and then incubated for 1 h in KRB buffer containing 3.3 mM or 16.7 mM glucose. The supernatants were collected and centrifuged (1000 rpm, 5 min) to measure insulin content using rat insulin ELISA kits (Linco Research, Inc., St. Charles, MO, USA) according to the kit's instructions.