Fitc conjugated anti ki67

Blood samples were collected into ACD (citric acid, citrate, dextrose) tubes before the first pulse of MP (baseline or day 0) and the following days until the patient’s discharge from the hospital. We were unable to collect blood samples at day 3 and day 8 for the patients discharged from the hospital before this time point. Whole peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll gradient (Eurobio, Les Ullis, France) and analyzed by flow cytometry (FACS Canto II, BD Bioscience). PBMCs were surface-stained with monoclonal antibodies: PerCP-conjugated-anti-CD4, APC-H7-conjugated-anti-CD45RA, BV450-conjugated-anti CD127, PeCy7-conjugated-anti CD25 (all from BD bioscience). Cells were then fixed and permeabilized using a fix/perm buffer (eBioSciences) following the manufacturer’s instructions and then intracellularly stained with PE-conjugated-anti-FoxP3 (259D clone) and FITC-conjugated-anti-Ki67 (BD Bioscience). The FoxP3 expressing CD4⁺ subset phenotype was defined as previously shown [8 (link)]. Naïve Treg cells were defined as CD4⁺CD45RA⁺FoxP3^low cells (nTreg) and effector Treg cells were defined as CD4⁺CD45RA⁻FoxP3^high cells (eTreg), while FoxP3 expressing non-regulatory Treg CD4⁺ T cells were defined as CD4⁺ CD45RA⁻FoxP3^low cells (non-reg FoxP3⁺ T cells).

Frozen PBMCs were thawed, washed and stained with the following combination of monoclonal antibodies, Pacific Blue-conjugated anti-CD3 (Biolegend), PE-Cy7-conjugated anti-CD56 (BD pharmingen), APC-Cy7-conjugated anti-CD16, PE-conjugated anti-NKG2A (Beckman Coulter), APC-conjugated anti-CD158a/h (Biolegend), APC-conjugated anti-CD158b1/b2/j (Biolegend), APC-conjugated anti-KIR3DL1 (Biolegend), Alexa Fluor 488-conjugated anti-NKG2C (R&D Systems), APC-Cy7-conjugated anti-CD27 (BD pharmingen), and PerCP-Cy5.5 conjugated anti-CD57 (Biolegend) monoclonal antibody. LIVE/DEAD Fixable Aqua was used for dead cell exclusion in every phenotypic analysis. The stained cells were then washed, fixed and permeabilized by BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer's protocol. Subsequently, cells were washed and stained intracellularly with FITC-conjugated anti-Ki67 (BD Pharmingen) for 30 min at 4°C. Cells were washed and re-suspended in PBS prior to flow cytometric analysis. Cells were acquired on FACS LSR-II and LSR Fortessa (BD Biosciences) and FACS data was analyzed by FlowJo v10 (BD). Samples with <20% live lymphocytes were excluded from the analysis.

Anti-lamin-A/C (n-18), anti-p-ERK1/2, anti-PCNA, and PE-conjugated anti-lamin A/C were obtained from Santa Cruz Biotechnology. Alexa Fluor 488-conjugated anti-lamin-A/C was obtained from Cell Signaling. Anti-CD3, anti-CD28, fluorescein isothiocyanate (FITC)-conjugated-CD45.1, V450-conjugated anti-CD45.1, PerCPCy5.5-conjugated anti-CD45.1, PerCPCy5.5-conjugated anti-CD45.2, APC-conjugated anti-CD45.1, v450-conjugated anti-CD45.2, FITC-conjugated anti-CD45.2, APC-conjugated anti-IFNγ, and V450-conjugated anti-CD4 were from Tonbo Bioscience. PECy5-conjugated anti-CD45.1, PECy7-conjugated anti-CD45.1, PE-conjugated anti-Tbet, FITC-conjugate anti-CD8, APC-conjugated anti-CD69, APC-conjugated anti-CD25, PE-conjugated anti-T-bet, FITC-conjugated anti-Ki67, PE-conjugated anti-IL-4, and biotinylated antibodies against B220, CD19, MHCII, CD11c, CD11b, CD44, CD49b, IgM CD25, and CD8α were from BD Biosciences.