Anti cd19 apc

Peripheral blood lymphocytes were immunophenotyped by multicolour flow cytometry using the following antibodies: anti-CD3 Pacific Blue, anti-CD56 FITC, Anti-CD27 PE, anti-CD28 APC, anti-CD1d PE, anti-CD19 APC, anti-CD27 V450, anti-CD38 PerCP-Cy5.5 (all from BD Biosciences, San Jose, CA, USA) and anti-CD45RA PerCP-Cy5.5, anti-CD62L PE-Cy7, anti-CD5 PE-Cy7, anti-CD24 APC-eFluor 780 and anti-CD4 APC-eFluor 780 (from eBioscience, Inc. San Diego, CA, USA). Staining was performed on whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturer’s instructions. A minimum of 250,000 events were acquired for T cell panels and 500,000 events for B cell panels to ensure adequate capture of rare populations. Subsequent detailed analysis of lymphocyte sub-populations was performed on the gated lymphocyte population using FlowJo (Treestar, Inc., OR, USA). Absolute counts for the different lymphocyte populations were calculated per litre of blood, based on haematology laboratory reported total lymphocyte count.

Isolation of PBMCs, T cells, B cells and monocytes Whole blood (10 ml) was collected in EDTA collection tubes from each subject, and PBMCs were isolated by density-gradient centrifugation with Ficoll-Paque Premium (GE Healthcate), according to the instructions. For the subsets of PBMCs isolation, the fresh PBMCs were incubated for 15 min at 4°C with flurescentconjugated monoclonal antibodies: anti-CD3-PerCP-Cy5.5, anti- CD14-PE, anti-CD19-APC (all from BD Biosciences). Stained cells were sorted on a BD FACSAria III (BD Biosciences). T cells were identified as CD3t/CD19-. Monocytes were isolated if cells were CD14t/CD3-. B cells were collected if cells were CD19t/CD3-. The stained cells were sorted to >98% purity.

For synergy and dose-response determinations, CLL cells were treated in 96-well plates with 6 increasing concentrations of drug for ~18 or ~72 h. Concentration ranges were selected and optimized to ensure that the exponential phase of the dose-response curve was obtained for the majority of the samples. Ranges were 0–160 µM for BEN, 0–80 µM for IDE and CLB, 0–20 µM for IBR and 0–10 µM for FLU. Synergy experiments with DRB were performed using 3 or 4 increasing doses of drug for ~18 h with the concentration ranges 0–40 µM for BEN and IDE, and 0–20 µM for DRB. The concentration of DMSO was constant between samples. After 18 or 72 h incubation, cell death was determined by flow cytometry by annexin-V-FITC and 7AAD (BD Pharmingen, San Jose, CA, USA) [41 (link)]. Cells were stained with for 15 mins with AV/7AAD and analyzed by flow cytometry using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) with a 96-well plate adapter. Cells were considered alive when they were double-negative for AV and 7AAD. When CD19 and CD3 were analyzed in the non-CLL donor PBMCs, anti-CD19-APC or anti-CD3-APC (BD Pharmingen) were added in a triple stain with AV/7AAD. Isotype control antibodies were also run for CD19 (anti-mouse-IgG1κ, BD Pharmingen) and CD3 (anti-mouse-IgG2aκ, BD Pharmingen).

Pleural and Bronchoalveolar cells were preincubated with murine Fc block CD16/CD32 and then stained with the following rat anti-mouse antibodies: anti-F4/80-APC (1:200; eBioscience, clone BM8), anti-SiglecF-PE (1:200, BD Bioscience, clone E50-2440), anti-Ly6G-V450 (1:200, eBioscience, clone 1A8), anti-CD4-PE (1:200; BD Bioscience, clone RM4-5) and anti-CD19-APC (1:200; BD Bioscience, clone 1D3). Macrophages were further analysed using anti-CD169-FITC (1:200, Biolegend, clone 3D6.112) and CD206-PE-Cy7 (1:200, Biolegend clone C068C2). Fluorescence Minus One (FMO) controls were used for each group with a pool of cells of all groups. The samples were run on a FACSVerse flow cytometer (BD Biosciences) and analysed using FACSuite software. Doublets and debris were excluded. CD169 and CD206 expression is expressed as mean fluorescence intensity (MFI) normalized by FMO (MFI-FMO).

The following antibodies were used for flow cytometric analysis: Anti-Perforin-Alexa fluor 488, anti-CD28-allophycocyanin (APC), anti-CD19-APC, anti-CD3-APC, anti-CD56-APC, anti-CD3-APC-cyanin7 (Cy7), anti-CD8-APC-Cy7, anti-CD14-APC-Cy7, anti-CD14-fluorescein isothiocyanate (FITC), anti-HLA-DR-FITC, anti-CD16-R-phycoerythrin (PE), anti-GATA3-PE, anti-CD45RA-PE-cyanin 5 (Cy5), anti-CD4-PE-Cy5, anti-CD16-PE-Cy5, anti-CCR7-PE-Cy7, anti-CD4-PE-Cy7, anti-CD4-V450, anti-GranzymeB-V450, anti-CD8-V500, anti-CD3-V500 (all from BD Bioscience, Franklin Lakes, NJ), anti-CD57-FITC, anti-CD4-FITC, anti-CD85j-PE, anti-CX3CR1-PE, anti-T-bet-PE-Cy7, anti-Eomes-Peridinin chlorophyll (PerCP)-efluor710 (six from eBioscience, San Diego, CA), anti-HLA-DR-PE-Cy5, anti-IL-7Rα-V450, anti-CD57-V450 (three from BioLegend, San Diego, CA), anti-CX3CR1-FITC (MBL International Corporation, Woburn, MA). For intracellular staining of T cell lineage-specific transcription factors (T-bet, Gata3, and Eomes), granzyme B and perforin, PBMC were fixed and permeabilized with Fix/Perm buffer set (BioLegend). Stained cells were acquired by a BD LSRFortessa (BD bioscience) and analyzed by using FlowJo software (ver. 9.0 or 10.0; Tree Star, OR).