Lsrii fortessa flow cytometer

Spleens were minced in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) and filtered through a 40-μm cell strainer to prepare single-cell suspensions. Tibias were dissected under sterile conditions to expose the bone marrow cavity, which was then rinsed with RPMI 1640 medium. Cells were surface-stained with eFluor780-fixable viability dye (FVD) (eBioscience, Carlsbad, CA, USA), Pacific Blue-conjugated anti-CD90.2 (BioLegend, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-CD19 (BioLegend, San Diego, CA, USA), PerCP-Cy5.5-conjugated GL7 (BioLegend, San Diego, CA, USA), and/or PE-conjugated anti-CD138 (BD Biosciences, San Jose, CA, USA) antibodies. The cells were then fixed, permeabilized, and incubated with anti-p62 antibody (Abcam, Cambridge, UK) or isotype antibody (Thermo Fisher Scientific, San Diego, CA, USA) followed by an Alexa Fluor 488-conjugated secondary antibody (Invitrogen/Thermo Fisher Scientific, San Diego, CA, USA). Cells were analyzed using a BD LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA).

Staining assays were performed to assess the intracellular expression levels of PTEN and USP18. The cellular permeabilization was done using 0.25% (W/V) saponine in PBS for 30 minutes at room temperature. The following multi-parameter antibody cocktail was used: anti-CD3-BB515, anti-CD4-BV605, anti-CD45RA-APC-Cy7, anti-PTEN-APC and anti-USP18-Alexa Fluor700. Of note, anti-USP18 IgG₁κ Abs was conjugated to Alexa700 dye using the Zenon mouse IgG₁ labeling kit (Life Technologies Inc.) according to the manufacturer’ protocol. The viability marker 7-AAD was used to exclude dead cells from analyses. BD LSRII Fortessa flow cytometer (BD) was used to collect the data which were analyzed using the DIVA software. Once again, we titrated all antibodies and washed the cells multiple times during the staining protocol to minimize all background fluorescences.

Ramos, SUDHL4, SUDHL1, SR-786, and U937 cells (2.5 × 10⁵ cells/ml) were treated with HDL NPs (50 nM) or PBS for 24, 48, or 72 h. Following treatment, C11-BODIPY (1 μM final concentration; Thermo Fisher Scientific) was added to each well and the cells were incubated for 30 min at 37 °C, 5% CO₂. The cells were then washed twice with 1 X PBS, resuspended in ice-cold FACS buffer and C11-BODIPY fluorescence in the FITC channel quantified using the BD LSR II Fortessa flow cytometer. Data were analyzed using the FCS Express software.

U2OS cells were synchronized in 5 µM l-mimosine (Sigma, M0253) for 24 h. After UV-C irradiation, the cells were left to recover for 6 or 18 h with the addition of 10 µM EdU (Invitrogen, C10337) for the final 30-min incubation. The cells were collected and fixed in 4% (w/v) paraformaldehyde (pH 8.0) for 15 min at room temperature. After permeabilization in 1× saponin buffer (Invitrogen, C10337) for 15 min at room temperature, the Click-iT reaction was performed according to the manufacturer’s protocol (Invitrogen). Thereafter, samples were incubated with 0.1 mg/mL RNase A (Thermo Scientific) for 30 min at 37 °C and the DNA was stained with 25 µg/mL propidium iodide (PI, Invitrogen) for 5 min. Using the BD LSR II Fortessa flow cytometer (10,000 cells per sample), PI signals were plotted against Alexa-488. Cell cycle distributions were evaluated using the FlowJo v.10.9 software.

Leukocytes were extracted from murine spleen, salivary glands and lungs as described previously [13 (link),60 (link)]. For CD4⁺ functional responses, isolated leukocytes were stimulated for 2 hours with 3 μg/ml m09, (GYLYIYPSAGNSFDL), M25 (NHLYETPISATAMVI), m139 (TRPYRYPRVCDASLS), and m142 (RSRYLTAAAVTAVLQ) MCMV MHCII peptides (Genscript). Brefeldin A (Sigma) was then added and cells incubated for a further 4 hours. For CD8⁺ functional responses, leukocytes were stimulated with 2 μg/ml m139, IE3, M38, and M45 MCMV MHCI peptides in the presence of anti-mouse CD107a-FITC (Biolegend) with monensin (BD Biosciences) and Brefeldin A for 6 hours. Cells were subsequently stained with Zombie Aqua fixable viability dye (Biolegend) or LIVE/DEAD-Aqua (Life-Technologies), stained with anti-CD16/CD32 Fc-block (Biolegend) and then with either anti-CD4 Pacific-Blue or PercP, (clone RM4-5, Biolegend) or with anti-CD8 PercP (clone 53–6.7, Biolegend) and anti-CD262 (TRAILR DR5, clone MD5.1, eBioscience). Cells were fixed, saponin permeabilised and stained for anti-IFNγ FITC or Pacific-Blue (clone XMG1.2, Biolegend) and anti-IL-10 APC (clone JES5-16E3, eBioscience). Data was acquired using a BD FACSCantoII or a BD LSR II fortessa flow cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar).

To assess bacterial infection in macrophages, HMDMs were harvested after adding non-enzymatic cell dissociation solution (Sigma-Aldrich, Oakville, ON, Canada) for 30 mins at room temperature. Cells were washed with PBS and prepared in FACS tubes. For surface staining, we used the following specific monoclonal antibodies provided from BD Biosciences for phenotypic analysis of human HMDMs: anti-CD3 (BV605), anti-CD14 (BV650), anti-CD80 (PE Cy7), anti-CD209 (PE) and anti-CD64 (PE). Finally, the viability marker 7-aminoactinomycin D or 7-AAD (ThermoFisher Scientific, St. Laurent, QC, Canada) was used to exclude dead cells from analyses. Afterwards, cells were fixed with 4% paraformaldehyde solution and then washed. For data analysis, samples were analyzed by flow cytometry with a BD LSRII Fortessa flow cytometer and DIVA software (BD). Viable gated cell singlets were analyzed for each sample and the percentages of mCherry^high CFT073 containing CD14⁺ HMDMs were determined. For Imaging flow cytometry, the same procedures were applied while cells were prepared in Eppendorf tubes. For surface staining, we used CD14-V450 and CD80-APC H7 for phenotypic gating of HMDMs. Samples were acquired using the Image Stream X MKII flow cytometer and analyzed with IDEAS software (Amnis) and representative images of singlets CD14⁺ HMDMs were generated expressing GFP^high CFT073 bacterial cells.