Sytox green dead cell stain

The killing assay has been described in detail elsewhere (18 (link)). In brief, PBMCs were isolated from blood of healthy donors by density gradient centrifugation. NK cells were enriched using the EasySep™ Human NK Cell Isolation Kit (Stemcell Technologies). After overnight incubation in complete medium using low dose recombinant human IL-2 (100 U/ml, R&D systems), cells were adjusted to 0.625 x 10⁶ vc/ml. EGFR positive A431 cells or EGFR negative ExpiCHO™ cells were stained with CellTracker™ Deep Red Dye (ThermoFisher). Target cells were seeded into 384-well clear bottom microtiter plates (Greiner Bio-One) at 2500 cells/well in 20 µl volume and incubated for 3 h. Afterwards, NK cells were added at different E:T ratios (i.e. 1:1, 5:1, 10:1 and 20:1). BsAbs were added at concentrations as indicated. An EGFR targeting Fc immune effector silenced antibody derivative was utilized as negative control. SYTOX™ Green Dead Cell Stain (Invitrogen, 0.03 µM) was dispensed to the assay followed by plate incubation and on-line measurement for 24 h in the Incucyte^® system. Lysis was normalized to maximum lysis triggered by therapeutic antibody cetuximab or to target cells cultivated with 30 µM staurosporine (Merck Millipore). Overlay signals allowed for analysis of dead target cells only.

Cells were seeded in 12-well plate (Corning) at 4 × 10⁴ cells per well. About 18 h after cell seeding, cells were pre-treated with indicated compounds for the indicated time before further treatment or directly treated with indicated compounds at the indicated concentrations for the indicated time. 30 nM SYTOX green dead cell stain (Invitrogen, S34860) was added into plates and incubated for 1 h at 37 °C. At least three randomly chosen bright fields and fluorescence fields were captured by microscopy (Olympus, IX51). Living cells (without green stained) and dead cells (with green stained) were counted, and the cell death ratio was calculated by the number of dead cells/numbers of (living cells + dead cells).

SW620 and HT29 cells were seeded at 2.5 × 10⁵ cells/well in 24-well plates before MccE492 or control treatments (phosphate-buffered saline (PBS) or mock). MccE492 was added to each well using a final concentration of 0, 30, or 60 μg/mL, and the mixture was incubated for 24 h. After incubation, cells of each well were trypsinized, suspended in PBS, and divided into two cytometry tubes. The viability was measured by staining the cells with 30 nM of Sytox Green dead cell stain (Invitrogen, Catalog No. S34860) and incubated for 20 min at room temperature protected from light. After incubation, 50 μL of Liquid Counting Beads (BD, Catalog No. 335925) were added to quantify the number of cells. Viability assays were analyzed by flow cytometry using a FACScan (Becton Dickinson) with BD FACSDiva™ Software.

Cell death was assayed via the Essen IncuCyte Live-Cell imaging system (Sartorius). Ten thousand K562 cells per well were seeded in black 96-well plates (Corning, 3904). Media containing a final concentration of 15 nM SYTOX-Green Dead Cell Stain (Invitrogen, S34860) and C6-Ceramide (d18:1/6:0) (Enzo, BML-SL110) at various concentrations was added to produce a final cell density of 500,000 cells/ml. Plates were carefully transferred to the IncuCyte system (kept at 37°C with 5% CO₂) and imaged for 24 h. Three images per well were captured in the green and brightfield channels every hour for the treatment period. The Sartorius image analysis software outputs the number of green objects (SYTOX-Green positive, i.e. dead cells) as well as the total number of objects (brightfield segmentation). For each C6-Cer concentration, the ratio of dead objects over total objects was plotted over the 24 h imaging cycle. From this, Prism (Graphpad) was used to calculate the area under the curve (AUC), plotted as function of C6-Cer dosage and mathematically determine the EC₅₀.

HCC cells were exposed to 21 and 1% O₂ for 5 days. Cells were collected by trypsinization and incubated with 30 nM SYTOX™ Green Dead Cell Stain (S34860, Invitrogen) for 40 min in the dark. Dead cell populations were analyzed by flow cytometry by BD LSRFortessa^TM flow cytometer (BD Biosciences) and the percentage of dead cell (SYTOX Green+, SG+) was calculated using FlowJo v10.7 software (FlowJo, LLC).

Proliferation assays were performed in serum-free AIM-V medium (Thermo Fisher Scientific) supplemented with penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Whole mouse splenocytes and human PBMCs were plated at a density of 5 × 10⁶ cells per well in a Cepallet W-type 24-well microplate (DIC) and cultured for 4 d with or without mouse/human M-CSF (Peprotech). Surviving macrophages that adhered to 24-well plates were detached by lowering the temperature on ice. The collected cells were incubated with both LIVE/DEAD fixable aqua fluorescent reactive dye (Invitrogen) and SYTOX Green dead cell stain (Invitrogen) in 1× PBS for dead cell staining. Whole mouse splenocytes and human PBMCs were stained with CD11b/Ly6G/NK1.1/Ly6C/Fcgr4 and CD11b/HLA-DR/CD14/CD16 after blocking antibody treatment, respectively. The number of live macrophages was estimated using flow-count fluorospheres (Beckman Coulter) and flow cytometry.
Sorted Ly6C^low and Ly6C^high monocytes were plated on 96-well plates (BD Falcon) at 2 × 10⁴ cells/well and cultured for 4 d with or without mouse M-CSF. The surviving macrophages were detected by the CellTiter-Glo 2.0 Cell Viability Assay (Promega) following the manufacturer’s protocol and the macrophage number was estimated by comparing with FACS analyses to the control samples whose monocyte numbers were counted.