Anti cd3 fitc 17a2

Single-cell suspensions of NALT cells were stained for 30 min at 4°C with appropriate combinations of fluorochrome-conjugated maternal antibodies in the presence of 0.2% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde in PBS. Cells were stained for surface markers with anti-CD284 (TLR4)-PE-Cyanine7 (SA15-21, Biolegend) or anti-CD284 (TLR4)-PE (SA15-21, Biolegend), anti-CD11c-PE (N418, eBioscience), or anti-CD11c-PerCP-Cyanine5.5 (N418, eBioscience), anti-CD19-APC (6D5, Biolegend), anti-CD3-FITC (17A2, eBioscience), anti-CD11b-PE-Cyanine7 (M1/70, eBioscience), anti-Ly-6G/Ly-6C (Gr-1)-APC (RB6-8C5, Biolegend), anti-F4/80-PE (BM8, eBioscience), and anti-CD282 (TLR2)-PE (CB225, Biolegend) as needed. For intracellular staining, fixed cells were permeabilized and stained in saponin (0.1% in PBS; Sigma) with anti-CD289 (TLR9)-FITC (M9.D6, eBioscience) and anti-CD283 (TLR3)-PE (11F8, Biolegend) in the presence of 1.5% BSA. Samples were analyzed using a FACSCanto flow cytometer (BD Biosciences) and FlowJo software (Treestar, Ashland, OR).

To deplete CD8⁺ T cells, the anti-CD8 monoclonal antibody (mAb) was prepared using the hybridoma clones in vivo in BALB/c nude mice. C57BL/6 J mice were intraperitoneally injected with 500 mg of the anti-CD8 mAb for 3 consecutive days (days -8, -7, and -6) and day -3 before day 0 when leukemia cells were subcutaneously inoculated and every 3 days since day 0 [8 (link)–10 (link)]. On day -2, CD8⁺ T cell depletion was verified by the flow cytometry (FCM) analysis of peripheral blood mononuclear cells (PBMCs) collected from the tail vein, and the analysis was conducted every 7 days since day 0 to confirm the sustained depletion of CD8⁺ T cells. For the analysis, T cells were stained with the anti-CD8-Alexa Fluor 647 (KT15, MBL, Nagoya, Japan), anti-CD3-FITC (17A2, eBioscience, San Diego, CA), anti-CD4-V500, (RPA-T4, BD Biosciences, Franklin Lakes, NJ), and anti-CD8a-V450 (53-6.7, BD Biosciences) mAbs. Tumor growth was monitored every 2 days.