Cd4 pacific blue clone okt4

Peripheral blood mononuclear cells were isolated from whole EDTA blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. Mononuclear cell preparations of intestinal biopsy and necropsy tissues were obtained by mechanical disruption and enzymatic dissociation followed by lymphocyte separation by centrifugation through Ficoll-Paque Plus (GE Healthcare), as described previously54 (link). Freshly isolated cells were immunophenotyped using the following antibody panel: CD4 Pacific Blue (clone OKT4; BioLegend; 1:50); CCR5 PE (clone 3A9; BD Biosciences; 1:20); CD28 ECD (clone CD28.2; Beckman Coulter, 1:20); CD95 PE-Cy5 (clone DX2; BD Biosciences; 1:10); CD8 PE-Cy7 (clone SK1: BD Biosciences; 1:100); CD38 APC (clone OK10; NIH Nonhuman Primate Reagent Resource; 1:25); CD3 APC-Cy7 (clone SP34-2; BD Biosciences; 1:100); and Ki67 FITC (clone B56; BD Biosciences; 1:20). Approximately 100,000 CD3+ T cells were acquired for each sample and data analysis was performed using FCS Express software. All dilutions are from the manufacturer's stock and based on 100 μl total staining volume.

CD4-PacificBlue (clone OKT4; 1:100), CD8-FITC (clone RPA-T8; 1:100), CD10-APC/Cy7 (clone HI10a, 1:100), CD69-PE (clone FN50; 1:200), CD276-PE/Cy7 (clone MIH42; 1:100) and the respective isotype control antibodies were purchased from BioLegend (San Diego, USA). CD45-AmCyan (clone 2D1, 1:200) was purchased from BD Biosciences (Franklin Lakes, USA).
For flow cytometry-based lysis assays, 100,000 target cells were incubated in 96 well plates together with 100,000 PBMCs, blinatumomab at 1 ng/ml and different tyrosine kinase inhibitors at the indicated concentrations. After 3 days, flow cytometric analysis was performed. Nalm-6 cells were defined as CD45⁻CD10⁺, Nalm-16 as CD45⁻CD10⁺, SD-1 per cell size and as CD22⁺, TOM-1 as CD45⁻CD10⁺, T cells as CD45⁺CD4⁺ or CD45⁺CD8⁺, and activated subsets were identified using CD69 as surrogate marker. Absolute cell numbers were determined by the acquisition of equal amounts of compensation particles (BD Biosciences) per sample, thus allowing for calculation of the absolute degree of tumor cell lysis. Binding of antibodies to Fc receptors was blocked with Flebogamma DIF (Grifols, Barcelona, Spain) at 50 μg/ml. Data acquisition was performed using a FACSCanto II (BD Biosciences). For data analysis, FlowJo_V10 software (Tree Star, Ashland, OR) was utilized.