CD20 mean fluorescence intensity (MFI) values were obtained before and on ibrutinib using peripheral blood mononuclear cells (PBMCs) prepared by density-gradient centrifugation (Lymphocyte Separation Media; ICN Biomedicals, Irvine, CA) and viably frozen in 90% fetal bovine serum (FBS), 10% dimethyl sulfoxide (Sigma, St. Louis, MO) in liquid nitrogen. Enumeration of anti-CD20 binding sites, antibody binding capacity (ABC), was performed on fresh PBMCs and fresh bone marrow cell suspensions in the clinical laboratory using the QuantiBrite system (BD Biosciences, Franklin Lakes, NJ) as previously described (25 ). Cells were analyzed on a FACS Canto II flow cytometer using FACS-DIVA 6.1.1, FCSExpress 4 (BD Biosciences) and FlowJo 10 software (TreeStar, Ashland, OR). In select experiments, the role of NF-κB on CD20 expression was measured using the NF-κB inhibitor Bay 11-7028 (Santa Cruz Biotechnology, Dallas, TX).
Quantibrite system
Manufactured by BD
The QuantiBrite system is a laboratory equipment designed for quantitative flow cytometry. It provides accurate measurement and analysis of fluorescent proteins and molecules in biological samples. The system utilizes standardized fluorescent beads to enable quantification of target analytes.
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2 protocols using quantibrite system
1
CD20 Expression Analysis in Ibrutinib-Treated Samples
2
Flow Cytometric Analysis of CCR5 Expression
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EDTA-anticoagulated whole blood obtained from each of the study participants (cohort 1) was stained within one hour of blood collection. Four antibody panels were used for each sample to assess CCR5 expression on T, B and natural killer (NK) cells as well as granulocytes and monocytes. Furthermore, HLA-DR was included as a marker in a fifth panel to assess the extent of cell activation (i.e., percentage of HLA-DR-expressing cells) – this was carried out on all controllers and a subset (16/22) of the HCs. The detailed staining/flow cytometry method has been previously described (29 (link)). Briefly, the CCR5 antibody used was conjugated to phycoerythrin (PE) at a ratio of 1:1, thereby allowing for CCR5 quantification, as the mean number of CCR5 molecules per cell (CCR5 density), in addition to the percentage of CCR5-expressing cells within a cell subset. Quantification was carried out using the QuantiBRITE system (BD BioSciences) which is a set of four precalibrated beads to calibrate the fluorescence 2 (FL2) axis in terms of PE molecules.
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