Anti cd8b clone h35 17.2

Splenocytes from male mice were cultured in 10% FCS/RPMI 1640 medium stimulated with 20 μg/mL of p210, a scrambled peptide, or equal volume of vehicle for 24 hours. CD8+ memory T‐cell profiling was performed on cultured splenocytes or bone marrow cells with or without peptide stimulation using CD44 (clone IM7), CD62L (clone MEL‐14; both from eBioscience), and CD8b antibodies. For intracellular cytokine staining, cells were cultured in Cellular Technology Limited (CTL) Test Medium and stimulated with p210, scrambled peptide, or vehicle for 48 hours. Cells were collected and subjected to fluorescent surface staining with monoclonal anti‐CD4 (clone GK1.5) and anti‐CD8b (clone H35‐17.2) antibodies (eBioscience) followed by fixation and permeabilization for fluorescent intracellular staining. Cells were then stained with monoclonal anti‐interferon‐γ (clone XMG1.2) and anti‐interleukin‐10 (IL‐10) (clone JES5‐16E3) antibodies (eBioscience) and data were acquired using a BD Fortessa flow cytometric apparatus.

Tumors (1 day after i.t. injection of PBS, iMSC, or iMSC/CCL19) were snap frozen in Optimal Cutting Temperature (OCT)compound (Sakura Finetek Japan Co.) by using liquid nitrogen. Sections were cut with a cryostat and fixed in cold 4% paraformaldehyde (Wako). Sections were stained with Rat isotype control mAbs (IgG2a: clone eBR2a, IgG2b: clone eB149/10H5, eBioscience), anti-CD3 (clone 17A2, TONBO), anti-CD4 (clone RM4-5, eBioscience), or anti-CD8α (clone 53-6.7, TONBO)+anti-CD8b (clone H35-17.2, eBioscience), and then with ImmPRESS Goat anti-Rat IgG peroxidase polymer detection kit (Vector Laboratories). mAbs were visualized with ImmPACT DAB peroxidase substrate (Vector Laboratories) and sections were counterstained with Hematoxyline QS (Vector Laboratories). For CD11c staining, endogenous biotin blocking with an avidin/biotin blocking kit (Vector Laboratories) was added after fixation. Sections were stained with biotinylated anti-CD11c (clone N418, AbD Serotec) or a Hamster isotype control IgG (clone eBio299Arm, eBioscience), biotinylated goat anti-Hamster IgG (Vector Laboratories), and then VECTASTAIN Elite ABC HRP Kit (Vector Laboratories), and visualized with ImmPACT DAB substrate.