Lsrfortessa x 20 flow cytometer

For intracellular staining of IFNγ and TNFα from NK and NKT cells, PBMCs were treated with Hsp70 peptide (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells in the presence of brefeldin A (BD Biosciences). Cells were then stained with antibodies against CD3, CD56, and CD16 conjugated to APC, FITC, and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with antibodies against IFNγ-PE (Beckman Coulter, Indianapolis, IN, USA) or TNFα-PE (Beckman Coulter) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FlowJo_V10 software (TreeStar).
For intracellular staining of IFNγ and TNFα from CD8+ T cells, PBMCs were treated with PepMix SARS-CoV-2 NCAP (JPT, Berlin, Germany) and CD3/CD49D (BD Biosciences) in the presence of brefeldin A (BD Biosciences). Cells were then stained with antibodies against CD3 and CD8 conjugated to APC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with antibodies against IFNγ-PE (Beckman Coulter, Indianapolis, IN, USA) or TNFα-PE (Beckman Coulter) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FlowJo_V10 software (TreeStar).

To clarify whether the HYAL4 protein is present on the cell surface, flow cytometry analysis was performed using anti-HYAL4 antibody. Rat skeletal muscle myoblasts, L6 cells, were obtained from JCRB, National Institute of Biomedical Innovation (Osaka, Japan), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 2.5% fetal calf serum under humidified conditions, and 5% CO₂–95% air at 37 °C.
Single-cell suspension from L6 cells was obtained by mechanical disruption with 100 mM phosphate-buffered saline (pH 7.4) (PBS) and straining through a 40 mm nylon mesh. Cells were counted and surface-stained with the primary antibody, anti-HYAL4 antibody (clone A-7), and the secondary antibody, Alexa Fluor 488-conjugated anti-mouse IgG(H + L) antibody (Abcam Inc., Cambridge, UK), on ice for 1 h each. After washing with PBS containing 0.1% bovine serum albumin three times, the cells were analyzed using a BD LSRFortessa^TM X-20 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

After isolation of the indicated tissues, cells were washed in PBS, and subsequently stained with fixable viability dye eFluor455UV (1:500 dilution) (eBiosciences) and mAb (Clone 93) (ThermoFisher) directed against the FcγRIII/II CD16/CD32 (0.5 µg mAb/10⁶ cells) for 20 min at 4 °C. Cells were washed in PBS/2% FBS supplemented with 0.1% w/v sodium azide and 10 mM EDTA, incubated with the relevant mAb for 20 min at 4 °C and washed again twice. When primary antibodies were biotin-coupled antibodies, cells were incubated with Streptavidin-PE/Cy5 (1:800) (BioLegend) for 20 min at 4 °C. Data were acquired with the BD LSRFortessa^TM X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo software version 10.5.3 (Tree Star Inc.). Cell sorting was carried out with the BD FACSAria™ III equipment. In all experiments, forward scatter (FSC)-H versus FSC-A was used to gate on singlets, with dead cells excluded using the fluorescence-coupled fixable viability dye. Lineage positive (CD3, CD19, NK1.1, Ly6G, and Ter119) cells were removed from further analysis.

Candida albicans was cultured in SOC broth before 50 μM bleogen pB1 was added and incubated for 24 h at 37°C with shaking at 350 rpm. Propidium iodide (1 μg/mL) was added and 10000 cells were evaluated using flow cytometry (BD LSRFortessa^TM X-20 flow cytometer, United States). All the experiments were repeated for three times.

To quantify multiple cytokines in ILF, we used the Cytometric Bead Array (cat. no. 558264, BD Biosciences), a flow cytometry application that enables the simultaneous measurement of the concentrations of multiple proteins in a small sample volume. We performed the procedure as per the manufacturer’s instructions. GM-CSF (cat. no. 558335), IFN-γ (cat. no. 558269), IL-4 (cat. no. 558272), IL-6 (cat. no. 558276), IL-10 (cat. no. 558274), IL-12p70 (cat. no. 558283), MCP-1 (cat. no. 558287), MIP-1α cat. no. 558325), TNF (cat. no. 558273), and VFGF (cat. no. 558336) were analyzed, as these proteins are commonly secreted by MDSCs and macrophages. All of the cytokine flexes were obtained from BD Biosciences. We analyzed 25 samples that contained confirmable and sufficient proteins using Closure samples. A total of 3×10³ events were collected using a BD LSRFortessa^TM X-20 flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). Protein concentrations were determined using BD CBA Analysis Software, version 1.0.2 (BD Biosciences). Protein levels were compared between CX3CR1^high and CX3CR1^low samples using the cut-off value for CX3CR1.