V500 conjugated anti cd3

In brief, cryopreserved peripheral blood mononuclear cell (PBMC) were thawed and surface-stained with the following antibodies to identify monocyte sub populations as previously described [17 (link)]: V500-conjugated anti-CD3, Qdot605-conjugated anti-CD14, Alexa700-conjugated anti-CD16, PE-Cy7-conjugated anti-CD56, PE-Cy7-conjugated anti-CD19, PE-Cy7-conjugated anti-CD20, APC-H7-conjugated HLA-DR monoclonal antibodies (mAbs), All antibodies were from BD Biosciences, except for Q605-conjugated anti-CD14 and yellow Live/Dead (Life Technologies). Stained PBMCs were acquired by flow cytometry, using a 4-laser custom BD-Fortessa instrument (Becton Dickinson) and analyzed with FlowJo software (Treestar Inc Ashland, OR) as previously described [17 (link)] (Fig 1).
The total monocyte count was calculated using white blood cell count (WBC) and percent monocyte values on the CBC conducted as part of entry evaluations on the same blood specimen as that utilized for flow cytometry in line with our previous reports [17 (link)].

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).