Pe labeled h 2kb ova257 264 pentamer

Ultrapure LPS Escherichia coli 0111:B4 was purchased from InvivoGen. Low endotoxin grade OVA (<0.01 EU/μg protein) was used for immunization (Hyglos). MHC-I H-2Kb immunodominant peptides OVA_257–264 (SIINFEKL) and TRP-2_180–188 (SVYDFFVWL) were ordered from GenScript with the TG-sequence at their N-termini followed with N-terminal extension from the native protein OVA (SGLEQLE)36 (link) allowing proteolytic processing to liberate the epitopes (NQEQVSPLSGLEQLESIINFEKL, NQEQVSPLSGLEQLESVYDFFVWL). PE-labeled H-2Kb/OVA_257–264 pentamer was purchased from ProImmune. Pentamer staining was performed according to the manufacturer's instructions.

Cells were washed and stained with surface antibodies in staining buffer [HBSS (Life Technologies) supplemented with 0.5 % bovine serum albumin]. Cell viability was determined by propidium iodide incorporation in staining buffer after surface antibody staining or with live/dead fixable cell viability reagent (Life Technologies) in PBS before antibody staining. Cells were stained with PE-labeled H-2Kb/OVA_257–264 pentamer (Proimmune, Oxford, UK) according to manufacturer’s instructions.
AccuCount cell counting beads (Spherotech, Lake Forest, IL, USA) were added to blood samples. Samples were acquired on CyAn ADP analyzer (Beckman Coulter, Brea, CA, USA), and data were analyzed with FlowJo software (v9.4; Tree Star, Ashland, OR, USA). Antibodies against mouse CD8, CD3, MHCII, B220, CD45, CD11b, Gr1, Ly6c, Ly6g, and CD11c were purchased from eBioscience or BioLegend (San Diego, CA, USA). Pacific orange-conjugated and Alexa Fluor 647-conjugated streptavidins were from Life Technologies.