Celltiter 96 non radioactive kit

All procedures concerning human TA biopsy and PBMC isolates were approved by the University of Florida Institutional Review Board (IRB) and conform to national standards. All patients gave written informed consent and only patients undergoing clinically indicated TA biopsy performed by a surgeon for suspected GCA were enrolled. TA biopsies were independently identified as GCA^pos (3 biopsies) or GCA^neg (7 biopsies) by standard pathologic indices. TA specimens were stored frozen at -80^°C in OCT medium with no fixative (Sakura Finetek USA, Inc, Torrance, CA, USA) prior to implant. CellTiter 96 Non-Radioactive kit (Promega Corp., Madison, WI, USA) was used with the minced tissue to measure the cell viability of the frozen arterial sections. Once the model was established the survival rate was high (~100%) and this high survival for animals with GCA^neg and GCA^pos TA transplants suggests that tissue integrity was preserved. PBMCs were isolated from a normal volunteer, collected and frozen at different times, and were pooled and enriched by Ficoll–Paque and followed by centrifugation (GE Healthcare Biosciences, PA, USA). Cell viability was evaluated by Trypan blue exclusion.

The Cell Titer^® 96 Non-Radioactive kit (Promega, Madison, WI, USA) was used to determine viable cell number based on the cellular conversion of a tetrazolium salt into a formazan product. The absorbance of the solubilized formazan product (directly proportional to the number of cells) was recorded using a 96-well plate reader daily over a 7- or 10-day period. Samples were seeded in triplicates.