Multiplex immunoassay kit

To identify the changes in islets, one pancreas biopsy was performed on the day of diabetes manifestation, immediately before the start of therapy, and another at the end of therapy as described previously [35 (link)]. The remaining pancreas comprising head and tail was collected for analyses 60 days and after anti-TCR combination with anti-TNF-α also 360 days (group 6) after the end of therapy including serum samples. Blood glucose concentrations were determined daily (Glucometer Elite®, Bayer, Leverkusen, Germany). Serum C-peptides were analysed with a rat-specific ELISA (Mercodia, Uppsala, Sweden) and serum cytokine protein concentrations with a multiplex immunoassay kit (Bio-Rad, Munich, Germany) [15 (link), 16 (link)].

Maternal venous blood samples (7.5 mL serum tube, S-Monovette, Sarstedt, Nümbrecht, Germany) were obtained in a fasting state early in the morning. The samples were centrifuged at 4000 rpm for 10 min at 4 °C in a Hettich MR20 centrifuge (Hettich Lab Technology, Tuttlingen, Germany), and the serum was pipetted into new tubes for storage at −80 °C until evaluation.
Leptin levels were measured using either a direct sandwich enzyme-linked immunosorbent assay (ELISA) kit from MERCK/Millipore, Darmstadt, Germany, a TECAN reader from Nano Quant infinite M200 Pro, Switzerland, or a multiplex immunoassay kit from Bio-Rad Laboratories (Hercules, CA, USA) in accordance with the manufacturer’s instructions.
Resistin levels were measured using a commercially available multiplex immunoassay kit (eBioscience, San Diego, CA, USA or Bio-Rad) in accordance with the manufacturer’s instructions.

Tissue specimens were fixed for microscopic analyses [29 (link)]. Blood glucose concentrations were determined daily (Glucometer Elite®, Bayer, Leverkusen, Germany). Serum C-peptide was analysed with a rat-specific ELISA (Mercodia, Uppsala, Sweden), and serum cytokine protein concentrations with a multiplex immunoassay kit (Bio-Rad) [25 (link), 29 (link)].