Anti mouse cd62l fitc

Three groups of 7-week-old female C57BL/6 mice were repetitively injected with PBS, 5 mg per kg of dNP2-dTomato or dNP2-ctCTLA-4, respectively, every other day for 14 days. The weight changes of the mice were daily monitored. At day 15, the mice were sacrificed and the morphologies of the spleen, liver and brain were carefully observed. The cytotoxicity of each protein against splenocytes and thymocytes was analysed using an Annexin V and 7-AAD staining kit (BD bioscience). The percentages of naive CD4 T cells in lymph nodes and spleen were analysed with isolated lymphocytes from each tissue after staining with 1/800 diluted anti-mouse CD4-PerCP-Cy5.5 (#45-0042), anti-mouse CD62L-FITC (#11-0621) and anti-mouse CD44-PE (#12-0441) FACS antibodies purchased from eBioscience. Liver toxicity of the proteins was analysed using an alanine aminotransferase activity assay kit (BioVision) and an aspartate aminotransferase activity assay kit (BioVision).

2×10⁶ PMBC were stained with PE H-2K^d AMQMLKETI MHC-I dextramer (Immudex) for 10 minutes in PBS with 5% FBS at room temperature. Anti-mouse CD3 Pac Blue, Anti-mouse CD8a PerCP, Anti-mouse CD127 APC (BD Bioscience) and Anti-mouse CD62L FITC (eBioscience) antibodies were added and incubated for 20 minutes at 4°C. Cells were washed twice with PBS 5% FBS then run immediately on a BD Biosciences LSR-Fortessa cell analyzer. CD3+CD8+ cells were analyzed for dextramer staining. The memory phenotype of dextramer positive CD8+ T cells was determined by analyzing the expression of CD62L and CD127 [47] (link). Central memory T cells were characterized as CD127+ CD62L+, effector memory CD127+ CD62L-, effector cells CD127- CD62L-, and transitional memory cells CD127- CD62L+.

The spleen of each mouse was collected in PBS and homogenized, following incubation in ACKlysis buffer to remove red blood cells. Then splenocytes were stored in cell saving medium (KeyGEN) at -80°C until analysis. For analysis, cells were rapidly recovered at 37°C and balanced in RPMI 1640. After centrifugation, the cells were re-suspended with culture medium and remained at 37°C incubator for 0.5 h for complete cells recovery. Then the cells were washed with PBS and stained with the diluted fluorochrome-conjugated monoclonal antibodies for 30 min within PBS containing 0.5% BSA on ice. The following antibodies were used: eBioscience Fixable Viability Dye eFluor 780, anti-mouse CD3-AF647 (Biolegend), anti-mouse CD4-R718 (BD Biosciences), anti-mouse CD8-BV510 (Biolegend), anti-mouse CD62L-FITC (eBioscience), anti-mouse CD44-Percp/Cy5.5 (BD Biosciences), anti-mouse CD19-BV510 (Biolegend), anti-mouse B220-Percp/Cy5.5 (Biolegend) and anti-mouse CD38-R718 (BD Biosciences), anti-mouse IgG1-FITC (Biolegend) and anti-mouse IgG2a-AF647 (Biolegend). All flow cytometry data were acquired on FACS Aria II flow cytometer (BD Biosciences) and analyzed with FlowJo software.