Ze5 flow cytometer

UAS-GFP x KMT2A-MLLT3 cells were transduced with retroviruses MSCV-Gal4 (DNA binding domain, DBD)—RXRA (ligand binding domain, LBD)—IRES—mCherry. Cells were treated, and after 48 h, GFP measured by a ZE5 Flow Cytometer (Biorad).

ICS was performed according to a standard protocol as previously described [24 (link)]. Briefly, splenocytes isolated from mice 2 × 10⁶ cells/well were plated in 24-well plates and stimulated with mix of VACV-specific peptides (SPYAAGYDL, SPGAAGYDL, VGPSNSPTF, KYGRLFNEI, GFIRSLQTI, and KYMWCYSQV) or with PMA (phorbol myristate acetate, 30 ng/mL) and ionomycin (1 µg/mL). Each peptide was added at the concentration of 20 µg/mL per well, and cells were incubated for 4 h at 37 °C in 5% CO₂ and for an additional 16 h with Brefeldin A (5 μg/mL, GolgiPlug BD Biosciences). On the next day, cells were stained with pre-titrated anti-CD3 MCA500SBB700 (BIO-RAD) anti-CD8 FITC (BD Pharmingen, San Diego, CA, USA) and anti-CD4 PerCP (BD Pharmingen, San Diego, CA, USA), fixed, and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Cells were then stained for intracellular cytokine detection with anti-IFN-γ APC and PE Rat Anti-Mouse IL-2 (BD Pharmingen, San Diego, CA, USA). Samples were analyzed on a ZE5 flow cytometer (Bio-Rad). Data were presented as the median and range of variation. Statistical analysis was performed using GraphPad Prism 8.0.1 software. The confidence level was calculated using the nonparametric Mann–Whitney U-test and one-way Kruskal–Wallis analysis of variance.

A ZE5 flow cytometer (Bio-Rad, Hercules, CA, USA) was used to measure the forward scattering and fluorescence of microbial cells. These parameters were acquired as pulse height signals for 10,000 events at a rate of 1 μL per second and at a fluorescence wavelength of 647 nm. Prior to analysis, quality control was performed using ZE-series QC beads (Bio-Rad, #12004403). The instrument tubing was cleaned by sequentially using 1% bleach, 70% ethanol, and sterile PBS. Data were acquired using the Everest software package v1.4.

Selection of cross-reactive plasma against group 1 and group 2 IAV NAs and the evaluation of the breadth of the best FNI mAbs were performed by binding to NA proteins expressed on the cell surface by flow cytometry. NA expressing ExpiCHO-S or Expi293F cells were harvested, washed twice in FACS buffer (PBS supplemented with 2% FBS and 2 mM of ethylenediaminetetraacetic acid), counted and distributed into 96-well U-bottom plates (Corning). The sera or mAbs were serially diluted and incubated with the cells for 45 min on ice and then washed in FACS buffer. Alexa Fluor 647-labelled goat anti-human IgG secondary antibody (Jackson Immunoresearch) was added at 1 µg ml⁻¹ to the cells, following 20 min of incubation on ice. Cells were then washed with FACS buffer and analysed on a ZE5 Flow Cytometer (Bio-Rad). Data were normalized to the NA-positive cell fractions and analysed with FlowJo software.

H9C2 cardiomyoblasts were transduced with adenoviral MFN2 constructs as described. Then, 72 hr later cells were co-stained with MitoSOX Red (5 μM for 10 min at 37°C) and MitoTracker Green FM 200 nM (Thermo Fisher, Cat# M36008 and #M7514, respectively). Fluorescence was analyzed on a ZE5 Flow Cytometer (Bio-Rad). Data were analyzed with FlowJo software version 10 and are presented as mean fluorescence intensity of five independent experiments.

Cells were plated in six‐well plates tissue culture plates (3516, Corning) and allowed to adhere overnight. Cells were treated with Vehicle (0.05% DMSO), CGM097 (200 nM), OTX015 (5 µM), or a combination of both drugs at their respective doses. Twenty‐four hours after treatment, cells were harvested, fixed/permeablized, and labeled with FxCycle PI/RNAse Staining Solution (F10797, Thermo Fisher Scientific), as per the manufacturer's protocol. After labeling, cell cycle was analyzed by measuring DNA content, using a ZE5 flow cytometer (Software v2.3.03.0, Bio‐Rad). After analysis on the flow cytometer, FCS files were exported and analyzed using FlowJo software v10.5.3 (FlowJo LLC, Ashland, OR). G1 cell cycle results were tested for statistical significance with a one‐way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism v.5 software (GraphPad Software Inc). The gating strategy used in the cell cycle analysis is presented in Figure S2.