Winlist software

Cells were treated with the aforementioned siRNA for 48 h. Cells were then isolated and stained with phycoerythrin (PE) Annexin V and 7-aminoactinomycin D (7-AAD; BD Biosciences, Franklin Lakes, NJ, USA). Cells were analyzed using FACS in order to detect the fluorescence of PE Annexin V positive cells. The proportion of PE Annexin V positive cells in the siRNA-treated population was determined using the Super-Enhanced DMax method and the WinList software (Verity Software House Inc., Topsham, ME, USA).

HCT116 and HT-29 cells (1×10⁶/plate) were seeded on 100 mm dishes and cultured at 37°C overnight. The next day, cells were incubated with either MLN0905 or DMSO for the indicated times. Adherent and floating cells were harvested and fixed in 70% ethanol and stored overnight at -20°C. Cells were centrifuged and cell pellets washed with 0.5% BSA in PBS and then stained with γH2AX antibody (Millipore Corporation, Billerica, MA) per the manufacturer's instructions and incubated overnight at 4°C. Pellets were then washed and stained with the Alexa-488 labeled secondary antibody (Molecular Probes, Life Technologies Corporation, Bedford, MA) diluted 1∶150 and incubated for 30 minutes at room temperature. Pellets were washed and re-suspended in propidium iodide and RNaseA in PBS. Cell-cycle distributions were determined using flow cytometry (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ) and analyzed using Winlist software (Verity Software House, Topsham, ME).

DNA was extracted from SKBR3 treated cells as previously described [73 (link)], with some modifications. The cells were detached from the flasks with trypsin and suspended in 0.5 ml of TE buffer (100 mM Tris-HCl, 100 mM EDTA pH 8). 50 µl of 10% SDS and 25 µl of 1 mg/ml Proteinase K were added to the cell suspension and incubated for 30 minutes at 55°C. Nucleic acids were extracted using phenol-chloroform, precipitated with 1/10V 3M Na-acetate and 2V absolute ethanol and resuspended in 100 µl TE buffer. The same quantity of extracted DNA was loaded on a 1% agarose gel prepared in 0.5X TE buffer containing gel red. Gel was visualized under UV light using a Bio-Rad Trans illuminator and photographed by using a Polaroid camera.
Apoptosis of SKBR3 cells after treatment with IC₅₀ of AgNPs-EPS for 24 h was analyzed by flow cytometry essentially as described by Riccardi [74 (link)]. Briefly cells were fixed in 70% ethanol, washed in PBS and resuspended in DNA extraction buffer (200 mM Na2HPO4, 0.1% Triton X-100). After staining with Propidium Iodide (1µg/mL) for 30 min, fluorescence intensity was acquired in the FL2 channel by flow cytometry on a FACSCalibur flow cytometer (BD, New Jersey, USA). Data acquisition was performed with CellQuest Pro (BD) software, and analyzed with WinList software (Verity Software House, Topsham, USA).

Nbs reactivity to VAR2CSA–expressing IE was measured by flow cytometry. Approximately 100,000 late stage IE (50 µl at 2×10⁶ IE/ml) labelled with ethidium bromide were incubated with 50 µl Nbs (0,1 mg/ml). Nanobody binding was detected by 50 µl mouse-anti-penta-His Ab (Qiagen; 34660 diluted 1∶100 in PBS) and a FITC-labelled anti-mouse Ab (Vector: FI-2000; diluted 1∶200 in PBS). Each labelling step was conducted for 30 min at 4°C. As a negative control, IE were incubated with the secondary and tertiary antibody only. Data from 5,000 IE were collected on a FC500 flow cytometer (Beckman Coulter). The mean FITC fluorescence intensity was determined using Winlist Software (Verity Software House).

The percentage of viable, apoptotic and necrotic cells was determined using annexin-V/PI staining for flow cytometry. Cells were trypsinized and washed twice using cell staining buffer (BioLegend, San Diego, CA, USA) and stained in Annexin-V binding buffer (BioLegend) using 2.5 μL of Annexin-V-FITC (Immunotools, Friesoythe, Germany) and 2.5 μg/mL propidium iodide (Sigma-Aldrich). The percentage of necrotic, (early) apoptotic, and viable cells was measured afterwards using the BD FACS-Calibur (BD Biosciences, Franklin Jakes, NJ, USA) flow-cytometer and data were analyzed using Winlist software (Verity Software House, Topsham, ME, USA).

A minimum of 100,000 PBMC or LPMC were washed in fluorescence activated cell sorter (FACS) buffer (phosphate buffered saline (PBS) with 1 mmol/L EDTA and 0.02% sodium azide) and labelled on ice for 20 minutes with monoclonal antibodies at predetermined optimal concentrations. Details of the specific antibodies (including clone and conjugated fluorochromes) can be found in Supplementary Table 1. Cells were then washed twice (450g, 5minutes) in FACS buffer and subsequently fixed with 1% paraformaldehyde (1% PFA). Cells were acquired within 48 hours on a FACS Canto-II flow cytometer (Becton-Dickinson, UK). Data analysis was carried out using Winlist™ software (Verity Software House, Maine, USA). The proportion of cells expressing any given surface marker of interest was determined by comparing fluorescence to that of an isotype-matched control antibody. Flow-count fluorospheres™ (Beckman Coulter, UK) were also added to all samples to determine absolute numbers.