Facslyric

Cell surface expression levels of FcεRI, c-kit, TLR4, and ST2 were determined by flow cytometric analyses using FITC-labeled anti-mouse FcεRIα, APC-labeled anti-mouse CD117, PE-labeled anti-mouse CD284 (TLR4) (clone SA15-21, BioLegend), and FITC-labeled anti-mouse T1/ST2 (clone DJ8, BioLegend), respectively. For intracellular staining, a fixation buffer (#420801, BioLegend) and an intracellular staining perm wash buffer (10X) (#421002, BioLegend) were used according to the manufacturer’s instructions with PE-labeled anti-mouse FcεRIα. Brefeldin A (#420601) and Monensin (#420701) were purchased from BioLegend. Data collected using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Glandbach, Germany) or a FACSLyric (Becton Dickinson, Franklin Lakes, NJ, USA) were evaluated with FlowJo software (Tomy Digital Biology, Tokyo, Japan).

A flow cytometer was adjusted as described in previous work [19 (link)]. Shortly, fluorochrome stock solutions were prepared in proportions of 1:1 of SYTO^®9 (5 mM) and PI (20 mM). Staining of live (24 h culture) and dead (killed with 70% isopropanol) cells was performed by adding 2 mL of culture to 6 µL of dye working solutions. Then, the solutions were kept for 15 min at room temperature (20 ± 2 °C) and analyzed with a flow cytometer (FACSlyric, Beckton Dickinson, Franklin Lakes, NJ, USA). The flow cytometer’s excitation laser had a wavelength of 488 nm, and the fluorescence emissions were collected in the red and green channels. Two solutions (SYTO^®9-stained LCS and PI-stained DCS) were used for the identification of live and dead bacteria cells and for the most appropriate cytometric parameters to avoid interference from emission spectra. Logarithmic signal amplification was employed in combination with forward scatter, side scatter, and fluorescence.

Serological markers HBsAg, anti‐HBc, anti‐HBs, HCV antibody (Ab) and HIV antigen/antibody (Ag/Ab) were determined with the Abbott ARCHITECT. HLA serotyping was conducted with Becton Dickinson FACSCanto or FACSLyric.

Fluorochrome stock solutions were prepared with accordance to producer instructions by adding SYTO9 (5 mM) and PI (20 mM) in proportion 1:1 to a tube as a working solution. For live (LCS) and dead (DCS) cell suspensions, 1 mL of 24 h bacterial suspension was centrifuged at 10,000× g for 5 min and resuspended in 1 mL of PBS for LCS or 1 mL of 70% isopropanol for DCS. Staining was performed by adding 2 mL of LCS or DCS with 6 µL of working solutions. The mixtures were incubated for 15 min at room temperature and read with a flow cytometer (FACSlyric, Beckton Dickinson, Franklin Lakes, NJ, USA). The excitation laser of the flow cytometer had a wavelength of 488 nm and the fluorescence emissions were collected in the red and green channels. SYTO9-stained LCS and PI-stained DCS were used to locate appropriate bacterial populations and standardize the most appropriate cytometric parameters to avoid interference from emission spectra. Forward scatter, side scatter, and fluorescence were recorded with logarithmic signal amplification. First, the forward and side scatter enhancements were configured, followed by the green fluorescence enhancement to detect live bacteria, and finally the red fluorescence enhancement was combined to indicate dead bacteria.

Platelet aggregation tests in the presence of several agonists were measured with an optical platelet aggregometer (APACT 4004, ELITechGroup, Puteaux, France) according to the manufacturer's instructions. For activation tests, platelets were stimulated or not with different agonists for 15 min and then incubated with various fluorophore‐conjugated antibodies. Anti‐CD62P‐PE was purchased from Becton Biolegend. Anti‐CD41‐PC7 and anti‐CD63‐PE were purchased from Beckman Coulter. Samples were analyzed in an FACSLyric (Becton Dickinson) flow cytometer.

Annexin V-APC, TMRM staining: Apoptotic cell death was assessed as previously described [46 (link)]. Cells were harvested using trypsin/EDTA, resuspended in supernatant and washed in ice-cold PBS. Then, cell pellets were resuspended in 300 μL Annexin V-APC-binding buffer (PBS, 2.5 mM CaCl₂) supplemented with recombinant chicken Annexin V-APC (ImmunoTools, Friesoythe, Germany) and incubated for 10 min on ice. Subsequently, samples were analyzed using a FACS Lyric flow cytometer (Becton Dickinson, Heidelberg, Germany). To detect loss of mitochondrial membrane potential, cell pellets were resuspended in PBS supplemented with 2% FCS and 50 nM tetramethyl rhodamine (TMRM) (Merck, Darmstadt, Germany) of the potentiometric dye. Cells were incubated at 37 °C for 20 min and fluorescence was analyzed using a FACS Lyric flow cytometer. The proportion of TMRM^low and Annexin V-APC⁺ cells was calculated using FACS Suite software.

A FACSLyric™ (Becton Dickinson, USA) flow cytometer was employed for cytokine detection, and Beckman Coulter’s FC 500 series (Beckman Coulter, USA) was employed for CBC determination. The Alinity and ARCHITECT Systems were used for IgG levels against SARS-CoV-2, and the EUROIMMUN Analyzer I was used for the ELISA reader. The incubator, centrifuge, vortex mixer and automatic cell counter were from Thermo Fisher Scientific, Inc. (USA). Phosphate-buffered saline (PBS) with a pH of 7.4 was purchased from Sigma‒Aldrich (USA), heparin and EDTA tubes were purchased from Becton Dickinson (USA), and RPMI-1640 medium was purchased from Life Technologies (USA).