Anti cd62l percp cy5

To investigate the LN and spleen homing, the BALB/c mice were subcutaneously inoculated with CpG‐ODN^FAM, Neoantigen^Cy3, CpG‐ODN^FAM + Neoantigen^Cy3 mixture, or thiolated nano‐vaccine in the right groin for five times per 4 d, respectively. Afterward, the inguinal LNs were harvested, and imaged by ChemiDoc MP Imaging System from Bio‐Rad. Consequently, single cells isolated from LNs or spleen through collagenase D and DNase I pre‐treatment were collected and then stained with CD11c‐APC, CD80‐PE, and CD86‐PE‐Cy7 (eBioscience) for immune response analysis. The activated T cells that isolated from spleen and blood were stained with anti‐CD3‐APC, anti‐CD4‐FITC, anti‐CD8‐PE and analyzed by flow cytometry.
To analyze the memory T cells, the blood collected from the mice with thiolated nano‐vaccine treatment was diluted with PBS (blood volume: PBS volume = 1: 1) and subject to a density gradient centrifugation in Ficoll Paque TM PREMIUM sterile solution at 800 × g for 30 min at 4 °C. Afterward, the cells were harvested and then examined by flow cytometry after staining with anti‐CD4‐FITC, anti‐CD44‐PE‐Cy7, and anti‐CD62L‐PerCP‐Cy5.5 antibody or anti‐CD8‐PE, anti‐CD44‐PE‐Cy7, and anti‐CD62L‐PerCP‐Cy5.5 antibody (eBioscience).

Isolated mononuclear cells were stained with surface markers for 30 min at 4°C, and dead cells were excluded by staining Zombie Aqua Fixable viability dye (BioLegend). For intracellular staining, surface-stained cells were fixed and permeabilized with the Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific). The following fluorescent dye-labeled antibodies were purchased from BD Biosciences, BioLegend, and eBioscience: anti-CD4-BUV395 (1:400, GK1.5), anti-CD25-APC (1:400, PC61), anti-CD314 (NKG2D)-PE (1:200, CX5), anti-CD62L-PerCP-Cy5.5 (1:400, MEL-14), anti-CD103-APC (1:400), anti-CD44-AF700 (1:400, IM7), anti-TCRβ-APC-Cy7 (1:400, H57-597), anti-CD45.2-BV605 (1:200, 104), anti-CD8-BV650 (1:400, 53-6.7), anti-CD11b-BV711 (1:1,000, M1/70), anti-B220-BV711 (1:400, RA3-6B2), anti-CD357 (GITR)-PE-Cy7 (1:400, DTA-1), anti-CD339 (Jagged1)-PE (1:100, HMJ1-29), anti-Eomes-AF488 (1:200, Dan11mag), anti-T-bet-PE-Cy7 (1:400, 4B10), anti-FoxP3-PE-CF594 (1:400, MF23), anti-GATA3-PE (1:400, TWAJ), and anti-CD152 (CTLA-4)-PE-Texas Red (1:400, UC10-4F10-11). Stained cells were analyzed using BD LSR Fortessa, and data were analyzed using Flow Jo software (Tree Star).