To evaluate the efficacy of hASC exosomes in dermal wound healing, we performed the scratch assay, a widely used wound healing assay, in HDF. HDF cells were grown to confluency in 35 mm dishes with μ-Dish inserts (Ibidi solutions™, 81176) to make consistent and reproducible 500 μm gaps. Cells were treated with hASC exosomes, as described in each experiment, then the inserts were removed and cell migration was imaged on the Keyence BZx-810 microscope at 4× magnification. Images were taken at the same location saved into the Keyence BZx-810 microscope with images taken at 0 and 18 h or 0, 18, and 24 h, as indicated in the individual experimental setup. The open areas between lateral cell boundaries were quantified using Image J software with the plugin Wound_healing_size_tool.
Bz x810 microscope
Manufactured by Keyence
Sourced in Japan, United States, Germany
The BZ-X810 is a digital microscope that captures high-quality images and videos. It features a 16-megapixel CMOS camera, a motorized stage, and a range of objective lenses. The microscope can be used for various applications, including brightfield, phase contrast, and fluorescence imaging.
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Lab products found in correlation
710 confocal microscope, by Zeiss (6 mentions)
Sytox green, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Bz x800 analyzer software, by Keyence (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Cellevent senescence green detection kit, by Thermo Fisher Scientific (3 mentions)
Bz x800 analyzer, by Keyence (2 mentions)
Crystal violet, by Carl Roth (2 mentions)
Sirius red, by Polysciences (2 mentions)
Lithium carbonate, by Thermo Fisher Scientific (2 mentions)
Hhs128, by Merck Group (2 mentions)
Luxol fast blue (lfb), by Thermo Fisher Scientific (2 mentions)
Ab191181, by Abcam (2 mentions)
Ab22035, by Abcam (2 mentions)
Ab5790, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
130 protocols using bz x810 microscope
1
Evaluating hASC Exosomes in Dermal Wound Healing
2
NF-κB p65 Immunohistochemistry in Kidney Tissue
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Kidney tissues were fixed with 4% paraformaldehyde, deparaffinized and 5 µm sections cut. Low pH antigen retrieval was performed and tissue was stained for NF-κB p65 (D14E12 rabbit monoclonal, 1:800 dilution, Cell Signaling 8242S), envision+ rabbit DAB chromogen detection system (Dako) was used. Images were collected using Keyence BZ-X810 microscope.
3
Alizarin Red S Staining of Mouse Incisors
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Anteroposterior sections of P8 mouse incisor were deparaffinized using decreasing concentrations of xylene and ethanol, stained in freshly prepared 2% (w/v) Alizarin red S solution (pH 4.1–4.3) for 2–3 min, dehydrated in acetone followed by acetone-xylene solution (1:1), cleared in xylene, and mounted with a synthetic mounting medium (Dahl, 1952 (link)). Sections were observed using a Keyence BZX-810 microscope in bright-field mode.
4
Barcoded Antibody Staining of Tissue Sections
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Barcoded antibody staining of tissue sections mounted on cover slips was performed using a commercially available CODEX Staining Kit according to the manufacturer’s instructions for FFPE tissue (Akoya Biosciences) and as recorded for HuBMAP Lymphatic System TMC (10.17504/protocols.io.be9pjh5n)32 . Images were acquired at 20X magnification using a Keyence BZ-X810 microscope with a metal halide light source and filter cubes for DAPI (358), TRITC (550), CY5 (647), and CY7 (750). Typical images are 7×9 (3.77 × 3.58 mm) including acquisition of 17 Z-stack images at 1.5 μm pitch. Raw images were collected using the CODEX Processor software (version 1.30.0.12). Drift compensation, deconvolution, z-plane selection, and stitching were performed using the Cytokit software10 (link), using the Docker33 container found at https://hub.docker.com/r/eczech/cytokit (version ‘latest’ uploaded on Feb 5, 2020). The display lookup table (LUT) for all figures presented in this paper is linear and covers the full range of data.
5
Cell Migration Tracking after PKCδ Modulation
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Cells were grown to confluency in 35‐mm dishes with μ‐Dish inserts (Ibidi solutions™, 81176) to make consistent and reproducible 500μm gaps. PKCδVIII was overexpressed in T80 cells or expression was knocked down in OCC for 48 h. The inserts were then removed and imaged on the Keyence BZx‐810 microscope at 4x magnification. Images were taken at the same location saved into the Keyence BZx‐810 microscope with images taken at 0, 24, and 48 h. Analysis of done measuring empty area between cells in µm2 using the FastTrack A1 software (Ibidi solutions™).
6
Corpus Callosum Fluorescence Intensity Analysis
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Sections were stained by the method described above, then observed and photographed with a Keyence BZ X810 microscope. The captured images were imported into Image J, and the fluorescence intensity per area (300 µm*120 µm; indicated by dotted line) of the corpus callosum region of 3 sections for each individual was measured. Prior to statistical analysis, mean values were calculated for each group (n = 3 mice each) based on the measured values, and relative values were calculated using the mean value of control mice as 100%. Fluorescence intensity statistics results are shown as mean ± SEM.
7
Immunostaining of Keratinocyte YAP
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Keratinocytes were immunostained as described above using anti-YAP and Alexa antibodies. After observation using a BZ-X810 microscope (KEYENCE), YAP-derived intensity localized in nuclei was analyzed using a BZ-X800 analyzer (KEYENCE).
8
RBC Dynamics in Microfluidic Devices
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Microfluidic devices with four separate channels (46 µm tall, 100 µm wide, 4 mm long) were coated with laminin derived from human placenta (Sigma) for 2 h at room temperature58 (link). RBCs were isolated and resuspended in PBS to 0.2% hematocrit. Devices were perfused with RBC suspension via syringe pump (Harvard Apparatus). Images were acquired using a Keyence BZ-X810 microscope with a 20x/0.8 objective.
9
Nuclei Isolation from Frozen OSA Tissue
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Nuclei were isolated from 42 mg flash-frozen OSA tissue by following the nuclei isolation kit protocol from 10× Genomics (Pleasanton, CA, USA, CG000505 Rev A) with minor adjustments to enhance homogenization while retaining nuclear morphology. The Lysis buffer provided with the kit was diluted with phosphate-buffered saline (PBS) to 0.5 strength and the sample was briefly homogenized (1-2 s) using a bladed homogenizer on ice, followed by a 5-min incubation on ice. The nuclei isolation protocol from 10× Genomics was then followed according to the manufacturer's directions. Nuclei were visualized and counted using trypan blue (ThermoFisher Scientific, Waltham, MA, USA) and ViaStain acridine orange/propidium iodide (AO/PI) (PerkinElmer, Waltham, MA, USA) to determine quality and quantity using the Keyence BZ-X810 microscope with 100× oil-immersion objective. Nuclei were counted by hand using a hemocytometer in addition to using the Biorad TC20 automated cell counter (Biorad, Hercules, CA, USA) to determine the concentration for library preparation.
10
Adipocyte quantification in white adipose tissue
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Epididymal and inguinal subcutaneous WAT was fixed in 10% formalin neutral buffer (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) for 24 h at 4 °C and embedded in paraffin following the standard protocol. Sections (5 μm) were stained with hematoxylin and eosin for 10 and 5 min at room temperature, respectively, and at least 750 adipocytes in eWAT from each mouse were visualized using a BZ-X810 microscope (Keyence, Osaka, Japan) and enumerated using a hybrid cell count software (BZ-X800 Analyzer, ver. 1.1.2.4, Keyence).
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