Bz x800 series

Monocyte derived dendritic cell migration assay was performed in a Boyden chamber system (Millipore QCM 24-well, Temecula, CA, United States). mo-DCs were suspended in assay medium (serum-free RPMI 1640 containing 0.05% BSA). 800 μl of indicated sample was placed in the lower chamber. To prepare the testing samples in the lower chamber, RBC supernatants were filtered using 0.45 µm filter (Millipore) to remove RBCs after being pre-treated with LPS (50 μg/ml) for 48 h in culture (L-sup) or medium as control supernatant (C-sup). 400 µl of each supernatant was diluted in 800 µl assay medium in well of 24 well plate in the lower chamber. 200 µl of 2 × 10⁵ mo-DCs labeled with 0.1 μg/ml of Hoechst in assay medium was added into the upper chamber and then incubated for 6 h. Afterwards, the plate was put in a microplate shaker to shake orbitally for 10 s at 500 rpm to ensure that the cells in wells were processed uniformly. 200 μl of cells from the low chamber well was transferred to 96 well plate and shaken for 10 s at 500 rpm and then Hoechst positive cells were counted under a Keyence fluorescence microscope (BZ-X800 Series, Itasca, IL, United States). Values calculated by ImageJ represented the mean ± SD of at least three independent experiments.

Immunofluorescence analysis was performed as previously described [21 (link)]. The slides were microwaved in AR9 buffer (Opal-4 Color Manual IHC kit; PerkinElmer, Waltham, MA, USA) and cooled for 30 min. Sections were then incubated in Antibody Diluent/Blocking Buffer (Opal-4 Color Manual IHC kit; PerkinElmer) for 10 min at room temperature and then incubated with primary antibodies (listed above). Triple staining was performed with antibodies against IL-8 and SerpinE1 overnight at 4 °C and antibodies against CD206 for 2 h at room temperature. Samples were washed three times in TBST for 2 min each and then incubated in Polymer HRP (Ms + Rb) (Opal-4 Color Manual IHC kit; PerkinElmer). Samples were rinsed and then washed three times in TBST for 2 min each and then incubated in Opal Fluorophore working solution (TSA Plus System; PerkinElmer) for 10 min at room temperature. Samples were rinsed in TBST, microwaved in AR9 buffer, and then mounted with VECTASHIELD Mounting Medium For Fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA). Photomicrographs were obtained using a light microscope with a digital camera (BZ-X800 series; Keyence, Tokyo, Japan). The ratios of triple IL-8-, SerpinE1-, and CD206-positive cells in immune cells and tumor areas were quantified using the BZ-H4C and BZ-H4CM analytic applications.

CytoSelect 24-well wound healing assay (Cell Biolabs, Inc) was used to consistently measure cell migration and/or proliferation for wound closure between mGF derived from WT and CD73-deficient animals. Briefly, mGF were seeded (5 × 10⁵ cells/ml) in the presence of the wound healing inserts for 24 h. After confirmation that a monolayer of cells was formed, the inserts were removed generating a 0.9 mm “wound field”. Cells were left undisturbed and monitored for wound closure after 24 h. mGF were fixed and labeled with DAPI fluorescence to stain cell nuclei. The experiment was done in triplicate for each group and repeated at least 3 times to ensure reproducible results. Pictures were taken using a fluorescence microscope (Keyence BZ-X800 Series) in 4× objective lens and the wound closure was measured per the total fluorescence area using the Fiji software.

RNAscope (Advanced Cell Diagnostics, ACD; Hayward, CA) was performed using BaseScope Reagent Kit v2 – RED (323900) kit by using specific probes targeting human SNHG8 (NC_000004.12) according to the manufacturer’s protocol. 3ZZ probe named BA-Hs-SNHG8-O1-3zz-st targeting 2-133 of NC_000004.12:118278708-118279137 was used. BaseScope is a chromogenic assay: red chromogen was used for SNHG8 detection which can be seen under a fluorescent microscope in the Texas Red spectrum. HEK293-T cells were fixed with paraformaldehyde and washed with PBS. Slides were then hybridized with target probes and incubated in a HybEZ oven (ACD) for 2 h at 40 °C. Next, signals were amplified and generated with a BaseScope Detection Reagent Kit v2 – RED. Cells were then counterstained with DAPI. SNHG8 expression was scored as positive if staining was present in HEK293-T cells. For visualizing the slides stained with SNHG8, a Keyence microscope (BZ-X800 series, Keyence fluorescent microscope, Keyence, IL, USA) and confocal microscope (Zeiss LSM 980 with Airyscan 2, Zeiss, Germany) were used. Images were captured at 40X and 60X magnification. ImageJ (https://imagej.nih.gov/) was used to quantify the mean intensity of SNHG8. The freehand selections tool was used to mark the transfected cells in green channel and the measure tool was used to quantify the SNHG8 signal in the red channel.

Human aortic valve leaflets from donated hearts were frozen in OCT and sectioned at 8 μm thickness. Murine aortic roots containing valve leaflets were frozen in OCT and sectioned at a thickness of 5 μm. For each sample, a total of 5 sections were obtained at 100 μm intervals and used in the histological assessment. For immunofluorescence, slides were blocked with 4% BSA and 0.5% Triton-X 100 in PBS for 1 h, then primary antibody staining was performed in blocking solution overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature prior to staining with DAPI for 10 min. Slides were mounted using ProLong™ Diamond Antifade Mounting media (Thermo Fisher Scientific P36965) and imaged at 20X magnification with the slide imager (Keyence BZx-800 series). For the quantification of fluorescence staining in murine sections, positive signal acreage on each aortic valve was obtained via automated measurement using ImageJ and normalized to the valve leaflet area. For human tissue sections, CCN3, CD68 and DAPI positive cells were counted manually. CD68 positive cells were normalized to DAPI positive cells; and CCN3 positive cells were normalized to CD68 positive cells.