Pe conjugated anti ccr7

Splenocytes harvested from the immunized mice were incubated with the described Gag peptides, in the presence of brefeldin A (eBiosciences Inc, San Diego, CA) for 10 h, at 37°C. After incubation, cells were washed twice with FACS buffer (HBSS, supplemented 2% FCS, 1 mM Hepes, 0.1% NaN₃), and nonspecific binding was blocked by incubating cells with anti-FcγR antibody (BD Pharmingen) at 10 µg/mL for 15 min at 4°C. The splenocytes (1×10⁶ cells/well) were stained in duplicate with PerCP-conjugated rat anti-mouse CD4⁺ antibody, and/or PE-conjugated anti CCR7 (BD Pharmingen) at a dilution of 1∶100 for 30 min at 4°C. The cells were washed twice with FACS buffer and resuspended in 200 µL of Cytofix/Cytoperm solution at 4°C for 20 min. Cells were then washed twice with Perm/Wash solution and stained with APC-conjugated anti-IFN-γ, or APC-conjugated anti-IL-2 and PE-conjugated anti-TNF-α antibodies (BD Biosciences) diluted 1∶100. Events acquisition was performed with a FACScalibur flow cytometer instrument and data was analyzed with CellQuest software (BD Biosciences). A minimum of 50,000 events were analyzed.

Bone marrow mononuclear cells (BMMNCs) were separated using Lymphocyte Separation Medium (HaoYang, Tianjin, China). Lymphocyte subsets were quantified by flow cytometry using the following directly conjugated mouse anti-human monoclonal antibodies: V500-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD45RA, and PE-conjugated anti-CCR7 (BD Biosciences, San Jose, CA). After incubation, RBCs were lysed, and white blood cells (WBCs) were fixed with a lysing solution (BD Biosciences). As previously reported [17 (link), 25 (link), 35 (link)], effector T cells, naïve T cells, effector memory T cells, and central memory T cells were identified as CD45RA⁺CCR7⁻, CD45RA⁺CCR7⁺, CD45RA⁻CCR7⁻, and CD45RA⁻CCR7⁺, respectively. Multi-parameter flow cytometric analyses were performed using a BD LSRFortessa (Becton–Dickinson). Data were analyzed using BD Diva software (Becton–Dickinson).