Novocyte 2000 r flow cytometer

The isolated ASC-exosomes were captured and labeled with the MACSPlex Exosome kit, human (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s instructions. Briefly, 2 μg of ASC-exosomes were incubated overnight at 4 °C with 39 different capture beads. The captured exosomes were labeled with a mixture of APC-conjugated anti-CD9, -CD63 and -CD81 antibodies for 1 h at room temperature. The bead populations and APC intensities were analyzed by NovoCyte 2000 R Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and data were analyzed using the NovoExpress software (ACEA Biosciences). Background was corrected with median intensity of anti-CD9-, anti-CD63-, and anti-CD81-APC signals. Assays were run in triplicate on ten independent samples.

The isolated cASC-EVs were captured and labeled with Dynabeads, according to the manufacturer’s instructions. Briefly, 2 μg of cASC-EVs were incubated overnight at 4 °C with capture beads. The captured EVs were labeled with a mixture of APC-conjugated anti-CD81 antibodies for 1 h at room temperature. The bead populations and APC intensities were analyzed using a NovoCyte 2000 R Flow Cytometer (ACEA Biosciences, San Diego, CA, USA), and the data were analyzed using NovoExpress software (ACEA Biosciences). The background was corrected with the median intensity of the anti-IgG-APC signals. Assays were performed in triplicate for three independent samples.

To determine the specificity of the obtained nanoparticles, 1 × 10⁶ EMT6/P cells were resuspended in 300 μL PBS with 1% BSA and mixed with PLGA nanoparticles at various concentrations. The mixture was incubated for 15 min, then cells were washed from unbound nanoparticles by centrifugation at 100× g and analyzed on a Novocyte 2000R flow cytometer (ACEA Biosciences, San Diego, CA, USA; excitation laser 640 nm, emission filter 675/30 nm).

Tongues were dissected and placed in Z-Fix (Anatech Ltd; Battle Creek, MI), followed by 70 percent ethanol. Prior to fixing, the number of lesions/tongue were recorded and categorized as large or small. Fixed tongues were paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) or CD31 (iHisto, Inc; Salem, MA). Cervical lymph nodes and spleens were crushed through 100uM cell strainers, rinsed with balanced salt solution and stained for CD4-fluorescein isothiocyanate (clone GK1.5; Biolegend, San Diego, CA) and Foxp3-phycoerythrin (clone FJK-16S; ThermoFisher Scientific, Walthan, MA). Data was collected on a Novocyte 2000R flow cytometer (ACEA Biosciences; San Diego, CA) and analyzed using FlowJo (Ashland, OR) software.

To test for the effects of induced sigma factors on host sporulation, we diluted overnight B. subtilis cultures in fresh DSM (OD₆₀₀ = 0.05) and dispensed each culture into multiple wells of a 96-well plate that was then incubated in a BioTek Synergy H1 plate reader (37°C, fast and continuous shake setting). Under these conditions, we determined that cells enter stationary phase after approximately 4.5 h, marking the onset of sporulation. At this time, we induced expression of the cloned gene by adding IPTG (final concentration 1 mM) to half the cultures in the plate. We added water to the rest of the wells, which served as noninduced controls. At 24 h, we quantified the number of spores and vegetative cells in each well using a flow cytometry assay that distinguished spores from vegetative cells (nonspores) based on differential uptake of the nucleic acid stain SYBR green (97 (link)). We diluted each sample in Tris-EDTA buffer (pH 8) and then fixed the cells in 0.5% glutaraldehyde for 15 min at 4°C. We stained the fixed samples with SYBR green (20,000× dilution of commercial stock, Lonza) for 10 min at room temperature in the dark. We then enumerated cells using a volumetric NovoCyte 2000R flow cytometer (Acea; excitation, 488 nm; emission, 530/30 nm) and an automatic gating pipeline (see Fig. S6).