Lsrfortessa high parameter flow cytometer

Human DLBCL cells (HBL1, MD-901, U2932 and SUDHL4) transduced with pTRIPZ-miR-28 or pTRIPZ-scramble were counted, and 200,000 cells were seeded in duplicate on a 48-well plate in fresh medium containing doxycycline at 0.5 μg/ml to induce miRNA expression. Serial dilutions of ibrutinib (Selleck Chem) were prepared in vehicle (DMSO), and equal volumes of the diluted drug were added to cells to achieve the desired final concentration. At least two independent experiments were performed per cell line. The number of viable cells was determined 72 h after the start of ibrutinib treatment using CellTiter-Glo® reagent. Luminescence was measured with an Orion Plate Luminometer reader (Berthold). Luminescence values in medium-only wells were subtracted to yield relative luminescence units (RLU), and the data were normalized to DMSO-treated or ibrutinib-treated control cells. The percentage of apoptotic (Annexin V⁺ Dapi⁻) or dead (Dapi⁺) cells was measured 72 h after ibrutinib treatment by Annexin V (Biolegend, Cat#640932) and Dapi staining, and RFP expression was measured by flow cytometry in an LSRFortessa High-Parameter Flow Cytometer and analyzed with FlowJo V10.4.2 software.

DFBL-18689-V2 was acquired from the Public Repository of Xenografts (PRoXe). PDX cells (0.25–1 × 10⁶) were injected IV into 6–8-week-old NSG mice, and mice were sacrificed on day 26–31 after injection. Single-cell suspensions were obtained from spleens, and erythrocytes were lysed (ACK Lysing Buffer). Cells were stained with combinations of the following antibodies: anti-mouse CD45-APC (Cat#559864, clone 30-F11), anti-mouse CD45.1-V450 (Cat#560520, clone A20), anti-mouse CD45.2-V450 (Cat#560697, clone 104) anti-human IgM-APC (Cat#551062, clone G20-127), anti-human CD20-FITC (Cat#555622, clone 2H7), and anti-human CD19-APC (Cat#555415, clone HIB19) (all from BD Pharmigen). For proliferation analysis, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (eBiosience) and stained with anti-ki67 (Abcam; Cat#ab16667); staining was revealed with alexa fluor 647 goat anti-rabbit IgG (H + L) (L.T.) (ThermoFisher, A-21245). Live cells were detected with DAPI (Sigma-Aldrich) or with LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher, L34959). Labeled cells were acquired with a LSRFortessa High-Parameter Flow Cytometer and analyzed with FlowJo V10.4.2 software.