Cd44 alexafluor700

CD45.2-V500 and TNFα-APC-Cy7 were purchased from BD Biosciences (San Jose, California). KLRG1-FITC, Eomes-PE, CD107a-PE, CD107b-PE, CD27-PE, CD127-PE-Cy5, CD127-PerCP-Cy5.5, Tbet-PerCP-Cy5.5, IFNγ-PerCP-Cy5.5, Eomes-PErCP-efluor710, CD45.1-PECy7, KLRG1-PE-Cy7, Tbet-PE-Cy7, IRF4-AlexaFluor647, CD44-AlexaFluor700, CD62L-APC-eFluor780, CD44-eFluor450, KLRG1-eFluor450, IFNγ-eFluor450, CD90.2-APC-eFluor780, CD45.1-APC-eFluor780, IL-2-PerCP-Cy5.5 were purchased from eBioscience (San Deigo, California). CD8-PE-TexasRed, GranzymeB-PE, GranzymeB-APC, Live-Dead-Violet, Live-Dead-Aqua and goat-anti-rabbit IgG-AlexaFluor647 and -AlexaFluor488 were purchased from Life Technologies (Grand Island, New York). H2D^b-GP33 monomers were prepared at UMMS; LCMV-specific (H2D^b-NP396, H2D^b-GP276) and Influenza A PR8-OVA_I-specific (H2K^b-OVA257) monomers were obtained from the NIH Tetramer Core Facility (Atlanta, Georgia). Intracellular TCF1 staining was performed using rabbit-anti-mouse TCF1 (Cell Signaling Technology, Danvers, Massachusetts) followed by staining with goat-anti-rabbit secondary (Life Technologies). Samples were analyzed on an LSRII flow cytometer (Becton Dickinson), and data were analyzed using FlowJo (Tree Star).

Tetramer enrichment was performed as previously described (32 (link)). In brief, spleen and peripheral lymph nodes (inguinal, lumbar, mesenteric, cervical, axillary and brachial) were harvested and processed into a single cell suspension by passing through a 100μm strainer. Cells were washed in cold 1X HBSS (Cellgro) and resuspended with 4μg/mL of tetramer PE conjugated NFM:I-A^b and/or APC conjugated MOG:I-A^b (NIH tetramer core (33 (link), 34 (link))) in Fc block (heat killed mouse and rat serum, Sigma-Aldrich) at a volume 2× the pellet volume. After 1hr at room temperature, cells were wash in cold FACS wash (0.1% BSA, 0.05% NaN3, 1x PBS) and resuspend in 200μL FACS wash plus 50μL of anti-PE and/or anti-APC beads (Miltenyi Biotec) and incubated for 30 min. on ice. Cells were then washed and enriched on a LS column (Miltenyi Biotec). Unbound and column bound cell numbers were determined with AccuCheck microbeads (Invitrogen) alongside with cell surface marker characterization performed with flow cytometry (LSR II, BD) and FlowJo software (Treestar). Antibodies used included; CD3ε-FITC (145-2C11, BD Pharmingen), CD11b- PerCP -Cy5.5 (M1/70, BD Pharmingen), CD11c-PerCP-Cy5.5 (HL3, BD Pharmingen), CD19- PerCP -Cy5.5 (1D3, Tonbo Biosciences), CD4-Brilliant Violet 510 (RM4-5, BioLegend), CD8-Brilliant Violet 785 (53-6.7, BioLegend), CD44-Alexa Fluor 700 (IM7, eBioscience).