PBMCs were isolated using Ficoll-Amidotrizoaat (Pharmacy LUMC) gradient centrifugation according to the standard operating procedure of the Medical Oncology department of LUMC. Isolated PBMCs were carefully resuspended and 3 times washed in PBS (B. Braun, Melsungen, Germany). Samples were fixed in 1.5% formaldehyde and permealized in ice-cold pure methanol. Cells were washed 3 times in staining buffer (PBS with 5% bovine serum albumin (BSA, Sigma, St Louis, USA)) and stained for 30 min on ice with anti-CD45-PerCP-Cy5.5 (BD Bioscience, Breda, the Netherlands), clone 2D1 anti-CD3-PE (BD, clone SK7), anti-CD14-AF700 (BD, clone M5E2), anti-CD15-PE CF594 (BD, clone W6D3) and anti-γ-H2AX-AF488 (Biolegend, clone 2F3), followed by another washing step and resuspension in PBS. Per experiment we used 1,000,000 cells or more when available. The cell acquisition was performed immediately after the staining procedure on the flow cytometer (BD LSR Fortessa Flow Cytometer analyzer, BD Bioscience, Breda, The Netherlands) and data were analyzed using BD FACS Diva Software version 6.2. The CD45+ cells were gated, after which the CD3+ T-lymphocytes, CD3− non-T cells (also harboring B lymphocytes) or CD14+ CD15− monocytes were analyzed for the geomean (as measure for the intensity) of γ-H2AX (Supplementary Fig. 2 ).
Lsr fortessa flow cytometer analyzer
Manufactured by BD
The BD LSR Fortessa Flow Cytometer is a high-performance analytical instrument designed for multi-parameter analysis of cells and particles. It utilizes advanced flow cytometry technology to provide accurate and reliable data. The core function of the BD LSR Fortessa is to detect, identify, and analyze various cell types and characteristics within a sample.
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2 protocols using lsr fortessa flow cytometer analyzer
1
Peripheral Blood Mononuclear Cell Isolation and Analysis
2
Evaluating Etoposide and PST Effects on Hemocyte Populations
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Hemocytes at a cell density of 5 × 10 5 cells/well previously attached to glass slides were incubated for 3.5 h in SSW (control) or SSW containing 50 µM of Etoposide or SSW containing 0.08 µM of PSTs (STX, C1/C2 or GTX5). Hemocytes were then treated with trypsin buffer (trypsin 0.2%, EDTA 0.4%) for 30 min. Detached cells were fixed with 4% paraformaldehyde for 1 h in Eppendorf tubes and centrifuged for 10 min at 1000g (20 °C). Hemocyte pellets were re-suspended in PBS, centrifuged for 5 min at 600g and permeabilized with 0.01% Triton X-100 for 10 min. After a PBS wash and centrifugation (5 min, 600g, 20 °C), hemocytes were labeled with TMRNEL and DAPI as described in section 2.4.4. Hemocytes (10000 per sample) were sorted by size and granularity (blue) as described previously (Bachere et al. 2015) and the percentage of TMRNEL-labeled cells (red) per population was determined using a BD LSR Fortessa™ flow cytometer analyzer.
Flow cytometry experiments depend on an adhesion step to avoid hemocytes aggregation. Thus, analyses could only be conducted for adherent hemocytes populations such as hyalinocytes and blast-like cells. Unfortunately, the potential effects of PST on granulocytes, a non-adherent hemocyte population, could not be determined.
Flow cytometry experiments depend on an adhesion step to avoid hemocytes aggregation. Thus, analyses could only be conducted for adherent hemocytes populations such as hyalinocytes and blast-like cells. Unfortunately, the potential effects of PST on granulocytes, a non-adherent hemocyte population, could not be determined.
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