Cytoexpert software

Peyer’s patches (PP) were isolated from the small intestine and cells were extracted as previously described^{16 (link)}. Briefly, PP were finely cut and incubated for 40 min at room temperature with collagenase/DNase. Cells were then filtered through a 70 µm cell strainer and pelleted by centrifuging at 300×g, 4 °C during 5 min and then resuspended in FACS buffer (PBS, 2% FCS, 5 mM EDTA) and counted before antibody staining. For flow cytometry, cells were first incubated on ice in FACS buffer containing an anti-CD16/CD32 antibody to block the Fc receptor for 10 min and then incubated 30 min on ice in the dark with antibodies against the following surface markers : CD45-PE-eF610 (eBiosciences, clone 30-F11), CD19-PerCp-Cy5.5 (Biolegend, clone 6D5), CD3-PeCy7 (eBiosciences, clone 145-2C11), B220-AF647 (BD Biosciences, clone RA3-6B2), IgD-BV421 (BD Biosciences, clone 11-26c.2a), IgM-FITC (BD Bioscience, clone II/41) and IgA-PE (eBiosciences, clone mA-6E1). Cell viability was evaluated using Fixable Viability Dye eFluor 506 (eBiosciences). Cell acquisition was performed using a CytoFlex (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter).

All tissues were used as freshly prepared. After rats were anesthetized with 1% sodium pentobarbital, whole blood was collected from the abdominal aorta, and spleen and bone marrow were also harvested by mechanical dissociation, followed by RBC lysis. Suspensions were washed with phosphate-buffered saline (PBS) prior to cell surface staining with FITC anti-rat CD8a, PE anti-rat CD4, APC anti-rat CD3, Per CP/Cy5.5 anti-rat CD11b/c, FITC anti-rat CD103 (αE Integrin), PE anti-rat CD80, FITC anti-rat OX-62, FITC anti-rat CD335 NKp46, and rat anti-CD68 antibodies. Data were acquired and analyzed on a Cyto FLEX S using Cyto Expert software (Beckman Coulter).

Cell viability was evaluated by ViaKrome 808 Fixable Viability Dye (Beckman Coulter) according to manufacturer’s instructions. Cells were stained at 24 and 72 h after infection, fixed with 1% Paraformaldehyde (PFA) (Bio-Rad laboratories, Hercules, CA, United States) and washed 1x PBS. Data were recorded with a Cytoflex LX cytometer running CytoExpert Software (Beckman Coulter). Three independent experiments were performed. n.s.: not significant.

Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.). All statistical analyses and data plotting were performed using the Graphpad Prism software (v8.0, GraphPad Software Inc., U.S.A.). Statistical analysis between groups was conducted using the paired t-test, and p value of< 0.05 was considered significant.

Cells were gently detached using trypsin and rinsed in PBS. After removal of cell clumps (70 μm cell strainer), individual cells were resuspended into 500 μL of PBS-0.1% BSA. The cells were analyzed on a cytoFlex device (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter). For FACS, infected neurons were sorted using a MoFlo Astrios EQ device (Beckman Coulter). Negative cells (either not transfected or transduced) were used to set up the gates.

The characteristics of MSCs were identified by flow cytometry and the methods for identifying MSCs were described as previous methods [29 (link)]. CytoFLEX flow cytometers (Beckman Coulter) were used for flow cytometry, and CytoExpert software (Beckman Coulter) was used for data analysis. Anti-human CD90-FITC (Catalogue #IM1839U), anti-human CD19-FITC(Catalogue #A07768), anti-human CD11b-FITC(Catalogue #IM0530), anti-human HLA-DR-FITC(Catalogue #IM1638U), anti-human CD34-FITC (Catalogue #IM1870), anti-human CD45-FITC(Catalogue #AO7782), CD105-PE(Catalogue #B76299), and anti-human CD73-PE (Catalogue #B76299) (BECKMAN COULTER, Brea, CA, USA)were purchased from Beckman Coulter.

Cells were dissociated with Accutase (Thermo Fisher Scientific). The cells were fixed with 4% paraformaldehyde (Sigma) for 15 min and permeabilized with 0.5 % Triton X-100 for 10 min at room temperature. The cells were subsequently incubated with primary antibodies for 1 h at 4 °C, followed by a 30 min' incubation with the fluorochrome-labeled secondary antibodies at room temperature. For detection of cell death, cells were stained with 2 ug/mL propidium iodide (PI,Sigma) for 15 min in the dark at room temperature. Then the cells were analyzed using CytoFLEX LX and Cytoexpert software (Beckman, USA). Primary antibodies used in flow cytometry are in Table S4.