Fitc labeled rat anti mouse cd8

The frequency of OVA-specific CD3⁺ CD8⁺ T lymphocytes was measured on day 14 (post-prime vaccination) by tetramer staining and direct immunofluorescence as described previously by Karan et al. [55 (link)]. H-2Kb SIINFEKL class I iTAg^™ MHC Tetramer (Kb-OVA₂₅₇) labeled with phycoerythrin (PE) (MBLI, Woburn, MA) was used in this assay. Surface CD8 and CD3 were stained with fluorescein isothiocyanate (FITC)-labeled rat anti-mouse CD8 (eBioscience, San Diego, CA) and PE-Cy5-labeled hamster anti-mouse CD3 (eBioscience) antibodies, respectively. Samples were acquired using a BD FACScan flow cytometer (Becton, Dickinson, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, Ashland, OR). Results are expressed as percentage of total CD3⁺ CD8⁺ T lymphocytes in peripheral blood that were positive to tetramer staining.

Washed platelets were isolated from peripheral blood and CD8⁺ T cells were isolated from the spleen using an EasySep Mouse CD8⁺ T Cell Isolation Kit (StemCell Technologies). Platelets were identified by staining with FITC-labeled rat anti-mouse CD41 (BD Biosciences). CD8⁺ T cells were identified by staining with FITC-labeled rat anti-mouse CD8 (eBioscience, Inc.). PECAM-1 expression was evaluated by staining with PE-Cyanine7-labeled rat anti-mouse CD31 (eBioscience, Inc.). Data were acquired on an LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.).

Blood samples were collected via submandibular bleeding on day 14 post-prime. The frequency of OVA-specific CD3+ CD8+ T lymphocytes was determined by tetramer staining and direct immunofluorescence, as previously described [35 (link)]. The tetramer used was the H-2Kb SIINFEKL Class I iTAg™ MHC Tetramer (Kb-OVA257) labeled with PE (MBLI, Woburn, MA). Surface CD8 and CD3 were stained with FITC-labeled rat anti-mouse CD8 (eBioscience) and PE-Cy5-labeled hamster anti-mouse CD3 (eBioscience) antibodies, respectively. Samples were acquired using a FACScan flow cytometer and analyzed with FlowJo software. Results are expressed as percentage of total CD3+ CD8+ T lymphocytes in peripheral blood that were positive for tetramer staining.