Histopaque gradient

For islet isolation, the pancreas was injected with Liberase TM (Roche), digested at 37°C, and islets purified using a Histopaque gradient (Sigma-Aldrich), and handpicked as previously described (48 (link)). For secretion studies, isolated islets were cultured overnight in RPMI supplemented with 10% FBS. Following a 30-minute preincubation in Krebs-HEPES-bicarbonate buffer (KHB; 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 0.2 mM MgSO₄, 1.5 mM CaCl₂, 10 mM HEPES, 25 mM NaHCO₃) with 3 mM glucose, GSIS was assessed by static incubation of 6 size-matched islets in KHB with 3 mM or 16.7 mM glucose for 30 minutes at 37°C. Mature insulin secreted into the media and total mature insulin content of the islets were assayed by ELISA (Crystal Chem; no cross-reactivity with proinsulins).

To check the effect of MIF on the expression status of certain inflammation-associated genes in PBMCs, cells were isolated from hamster blood using the Histopaque gradient as suggested by the manufacturer (Sigma, USA). The isolated PBMCs were cultured in 10% RPMI media and treated with BSA (100 ng/ml), MaMIF (100 ng/ml)^{36 (link)–38 (link)}, Polymyxin B (30 μg/ml), MaMIF + Polymyxin B, ISO-1 ((S, R)-3-(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester) (100 μg/ml) or MaMIF + ISO-1 for 4 hours. Then cells were harvested in RLT buffer and processed for RNA isolation as described by the manufacturer (Qiagen, USA). RNA quantification was carried out using Nanodrop followed by cDNA synthesis^{11 (link)}. The expression levels of Tnf-α Il-6, Il-1β and Vegf, and were analyzed in all the cDNA samples through qPCR, by using gene-specific primers. Polymyxin B was used to neutralize any residual LPS present along with MaMIF protein.

Mouse islets were prepared as in Ravier and Rutter (2005) (link). Briefly, pancreata were perfused via the pancreatic duct with a NB8 collagenase solution (SERVA, Uetersen, Germany). Pancreata were digested at 37°C for 10 min and separated using a histopaque gradient (Sigma-Aldrich). Islets were handpicked under a microscope and cultured overnight in RPMI 1640 containing 11.1 mM glucose.