Whole blood from euthanized mice was collected by venipuncture directly into sodium citrate (3.2% w/v) at a ratio of 9:1 v/v. Platelet poor plasma was obtained from citrated blood after centrifugation and stored in −80o C.17 (link) Thawed plasma was recalcified and protimes measured on a KC4 coagulation analyzer (Trinity Biotech) after recalcification to 8.3 mM and mixing with thromboplastin (Dade Innovin®, Siemens Healthcare Diagnostics).
Quantification of antigen-specific cell numbers in peripheral blood was measured using a whole blood assay.18 (link) Blood was stained directly with fluorescent antibody cocktails along with PE-conjugated Kb- SIYRYYGL pentamers. AccuCheck fluorescent beads (Invitrogen) were added to each sample before lysing the red blood cells with BD FACS lysing solution (BD Biosciences) and samples analyzed on a BD LSRII flow cytometer. Cell concentrations were determined by comparing cellular events to bead events.
Quantification of antigen-specific cell numbers in peripheral blood was measured using a whole blood assay.18 (link) Blood was stained directly with fluorescent antibody cocktails along with PE-conjugated Kb- SIYRYYGL pentamers. AccuCheck fluorescent beads (Invitrogen) were added to each sample before lysing the red blood cells with BD FACS lysing solution (BD Biosciences) and samples analyzed on a BD LSRII flow cytometer. Cell concentrations were determined by comparing cellular events to bead events.