Facscalibur equipment

SV40 TAg-transformed or 3T9-immortalized MEFs, mouse monocytic FDMs, human monocytic U937 cells and human colon carcinoma HCT116 cells were infected with 10 moi of SFV or 50 moi of HSV-1 while shaking in DMEM plus 0.5% FCS. After 1 h at 37°C, viral infection was stopped and the cells incubated in DMEM plus 10% FCS until processed for further experiments. Alternatively, the cells were treated with 50 ng/ml FasL (kindly obtained from Pascal Schneider, Lausanne), 10 ng/ml TNFα (R&D Systems) or 500 ng/ml leucine zipper (lz) TRAIL (kindly provided by Henning Walczak, London). Apoptosis was quantified by annexin-V-GFP/propidium iodide (PI) FACS analysis using a FACS Calibur equipment from Becton Dickinson, and caspase-3/-7 activity was measured by the DEVDase assay as described [68 (link)]. Fluorescence was detected in the Fluoroskan Ascent equipment (Thermo Labsystems) and the relative fluorescence units (RFUs) were normalized to the protein concentration.

To evaluate toxicity of the antibodies and pharmacological inhibitors, cells were cultured in the presence or absence of anti-CD81, PD98059 (30μM), SB203580 (10μM) or SP600125 (10μM) for 72h. Then, the cells were stained with propidium iodide (PI, 50μg/ml, BD Bioscience) and analyzed by flow cytometry, using FACSCalibur equipment and CellQuest software. To further confirm the data, the viability of B cells cultured with anti-CD81 were also assessed by XTT assay (Sigma Aldrich), according to the manufacturer’s protocol; and the concentration of lactate dehydrogenase (LDH) in culture supernatant were evaluated, using LDH assay Kit (Doles, Goias, Brasil).

The cellular DNA was stained using the BD Cycletest™ Assay (BD Biosciences) and detected using a FACSCalibur™ equipment (BD Biosciences) using the CellQuest™ software. The DNA content was analyzed for 5000 cells. Percentages of cells in G0/G1, S and G2/M phases was established using the ModFit LT™ software (Supplementary Materials). The GFP was detected in HepaRG cells by fluorescence microscopy using Zeiss Inverted Microscope, and photographs were analyzed with the AxioVision Software. The quantification of GFP was carried out by flow cytometry: The cells were detached with trypsin and resuspended in William’s E medium containing FCS. The fluorescence intensity of 10,000 cells was analyzed with a Becton Dickinson le LSRFortessa™ X-20 (cytometry core facility of the Biology and Health Federative research structure Biosit, Rennes, France) to measure the fluorescence of the GFP positive cells. Flow cytometry data were analyzed using DIVA software (Becton Dikinson).

Blood serum was used for the quantification of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin 17A (IL-17A), and interleukin-10 (IL-10) by flow cytometry with FACS Calibur equipment (BD Biosciences, San Jose, CA, USA) using the BD™ Cytometric Bead Array (CBA) cytokine kit Mouse Th1/Th2/Th17 (BD Biosciences, San Jose, CA, USA) following the manufacturer’s recommendations.

H2A2-GFP was analyzed by wide-field fluorescence microscopy as reported before^{15 (link)}. Briefly, series of z-focal plane images (15–20 planes, 0.15–0.3 μm depth) were collected on a Leica DMI6000, using a 63×/1.30 immersion objective and an ultrasensitive DFC 350 digital camera, and processed with the AF6000 software (Leica). Scale bars in micrographs depict 5 μm; BF stands for bright field.
DNA content by flow cytometry analysis (FACS) was done as previously described using a BD FACScalibur equipment^{7 (link)}. An asynchronous culture of each strain growing at permissive temperature was used to calibrate the 1C and 2C peaks before reading the samples. Strains carrying the cdc15-2 and cdc14-1 alleles rendered a minor 2C peak at the G1 arrest. This peak corresponded to 10–20% of G1 samples and comprised cells responsive to alpha-factor but that remain attached as pairs (Figure S7). This phenotype likely stems from delayed septation at permissive temperatures. This minor 2C transposes into a 4C peak when cells are released from the G1 arrest, giving the false impression of re-replication.