Ctl immunospot s6 micro analyzer

Peptide reactive T cells were quantified using ELISPOT kits (Mabtech, Inc., Mariemont, OH). Briefly, plates were coated with capture anti-IFN-γ or anti-IL-17 antibodies. Splenocytes from C57BL/6, HLA-A2, K14-SCF, h3TA2 or Vitesse mice were cultured in RPMI1640 plus 10%FBS in presence of 10-30μg/ml mouse (FMDGTMSQV) and human (YMDGTMSQV) tyrosinase peptides (Pi Proteomic, LLC, Huntsville, AL) or PBS and plated at 5×10(5) cells/well. After 24h for IFN-γ or 48h for IL-17 detection, cytokine secreting cells were detected using biotinylated anti-cytokine antibodies, followed by horseradish peroxidase-conjugated streptavidin (Mabtech, Inc.) and 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Cytokine secreting cells were quantified using a CTL-ImmunoSpot S6 Micro Analyzer (Cellular Technology Ltd, Shaker Heights, OH). For intracellular cytokine staining, see above section

Patient PBMC were thawed and resuspend at 3×10⁶/ml in AIM-V^® AlbuMAX^® serum-free media supplemented with 1mM L-glutamine. The KTR responders were run in triplicate in media, test wells and Phytohemagglutinin (Sigma-Aldrich, St Louis, MO, USA) as a positive control. The MLR consisted of 3×10⁵ thawed KTR PBMCs plated on an anti IFN-γ antibody (2G1, Thermo-Scientific, Woburn, MA, USA) coated ELISPOT plate (Millipore, Bellerica, MA, USA) co-incubated with 4 or 5 B cells lines at 5×10⁴/per B cell line, 2 B cell lines per well (total of 1×10⁵ B cells/well), or with the 5^th B cell line at 1×10⁵ B cells/well, for 24h. These B cell lines had either 17 or 21 disparate antigens, with the most similar cell lines paired. After which, the plates were handled as per previously published protocol^{23 (link)}, with half the plates washed utilising an ELx50 Microplate strip washer (BioTek, Winooski, VT, USA).
The images captured with Cellular Technology, Ltd (CTL)-ImmunoSpot^® S6 Micro Analyzer (Cellular Technology, Ltd, Shaker Heights, OH, USA) with ImmunoCapture 6.4 software (Cellular Technology, Ltd). The spots counted using CTL ImmunoSpot 5.0 (Cellular Technology, Ltd) and quality controlled by inspecting every well and adjusting sensitivity. The IFN-γ ELISPOT plates were analysed at the Clinical Trials of Organ Transplantation (CTOT) center in Mount Sinai School of Medicine, New York.