The migration assay was performed in the Boyden chamber using cell culture inserts with a light-opaque polyethylene terephthalate of 8 μm microporous membrane (BD Falcon HTS FluoroBlokTM inserts, BD Biosciences). hBMSCs were dissociated with Accutase (Innovative cell technologies, San Diego, CA, USA), counted and seeded in the upper chamber (cell insert), and incubated for 24 h in different pH conditions. The cells were fixed with 4% PFA and washed with PBS, before nuclei staining and observation under a fluorescent microscope (Biozero BZ-X700, KEYENCE). The total number of migrated cells observed at the bottom of the chamber were counted in four different pictures taken per chamber/insert. Images of the cells were captured using fluorescence microscopy (Biozero BZ-X700, KEYENCE) and further binarized and counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Biozero bz x700
Manufactured by Keyence
Sourced in Japan
The Biozero BZ-X700 is a digital microscope designed for laboratory use. It features high-resolution imaging capabilities and advanced image analysis tools. The core function of the Biozero BZ-X700 is to provide users with detailed visual data and analysis of microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
Dm2500, by Leica (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Alexa fluor 546 labeled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Isolectin b4 ilb4, by Vector Laboratories (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Alexa fluor 488 or 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab38898, by Abcam (1 mentions)
Complete freund s adjuvant, by Chondrex (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Isotype igg, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti cd146 antibody, by Abcam (1 mentions)
15 protocols using biozero bz x700
1
Boyden Chamber Migration Assay for hBMSCs
2
Lung Morphology and Vascular Remodeling
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First for the morphological analysis of the lungs, the hearts and lungs were flushed with saline and then dissected (Zhang et al. 2017) . After that, the lungs were fixed with 4 % paraformaldehyde, embedded in paraffin, sectioned into 3-μm thick slices, and stained with Elastica van Gieson. Images were obtained using a digital microscope (Biozero BZ-X700; Keyence, Osaka, Japan). Pulmonary vascular remodeling was quantified by assessing the medial wall thickness. To determine the degree of medial wall thickness, 10 muscular arteries categorized <50 μm and ≥50 μm in diameter from each lung (n=6) were randomly outlined by an observer blinded to the mouse genotype or pharmacological treatment. The degree of medial wall thickness expressed as the ratio of medial area to cross sectional area (Medial/CSA) was analyzed using the ImageJ software (Ver. 1.48, NIH, USA).
3
Guard Cell Imaging and Oligosaccharide Treatment
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Rosette leaves were excised and gently mounted on a glass slide with the adaxial side upward using a medical adhesive (Hollister). Then, the adaxial epidermis and the mesophyll tissue were removed by a razor blade leaving lower epidermis intact on the slide. The lower epidermis was submerged in solution (5 mM KCl, 50 μM CaCl2, and 10 mM MES-Tris [pH 6.15]) at 22 °C in the light for 3 h resting. After staining of the epidermis with FDA (2 µg/mL), guard cells were imaged using a fluorescent microscope (Biozero BZ-X700; Keyence, Osaka, Japan) and then treated with oligosaccharides for 2 h, followed by imaging again. Inhibitors were added 30 min before oligosaccharide treatment. Only the guard cells stained by FDA before treatment were taken into account for the calculation of FDA staining rate, which was considered as the ratio of the number of FDA-stained guard cells after treatment to the number before. Propidium iodide at 10 µg/mL was used for staining. For FDA and PI double staining, guard cells were imaged using a fluorescent microscope (Leica DM2500; Leica Microsystems, Wetzlar, Germany)
Mesophyll protoplasts used for FDA staining were prepared according to ref. 79 (link). After treatment of (GlcN)8 for 2 h, mesophyll protoplasts were subjected to FDA staining.
Mesophyll protoplasts used for FDA staining were prepared according to ref. 79 (link). After treatment of (GlcN)8 for 2 h, mesophyll protoplasts were subjected to FDA staining.
4
Genetically Engineered B. subtilis Strain
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Genetically engineered construction on the basis of the pGFPAmy plasmid which contained a gene marking the green fluorescent protein GFPmut3 (excitation/emission-482/502 nm) was used for B. subtilis 26D recombination. Promotor Pveg of B. subtilis was amplificated by Pfu-polymerase using primers PvegF 5′-GGAGTTCTGAGAATTGGTATGCCTTAT-3′ and PvegR 5′- ACTACATTTATTGTACAACACGAGCCC-3′. pGFPamy was treated with endonuclease SmaI and ligated with Pveg [54 (link)]. Plasmid PGFPamyPveg was transformed into recipient strain B. subtilis 26D, giving ectopic insertion at the AmyE locus by a double cross-over recombination event (Figure 7 I). Bacteria were selected on LB medium containing 100 µg/mL chloramphenicol. Fluorescence was observed using the microscope Biozero BZ-X700 (“Keyence”, Japan) with the standard filter (Figure 7 II).
5
Quantifying Collagen Deposition and Nampt Expression
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Tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned into 4-µm-thick slices, and stained with Masson’s trichrome. Images were obtained with a digital microscope (Biozero BZ-X700; Keyence, Osaka, Japan), and collagen deposition was quantified by dividing the collagen deposition area by the total area. The analysis was performed using ImageJ version 1.45 software (NIH, Bethesda, MD, USA).
To evaluate Nampt expression, we performed immunohistochemistry. After deparaffinization and antigen activation, the sections were incubated with a rabbit anti-Nampt monoclonal antibody (LS-C137911; LifeSpan BioSciences, Seattle, WA, USA) at 4 °C overnight. Subsequently, the sections were thoroughly rinsed with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ab6721; Abcam, Cambridge, UK) at room temperature for 1 h. After rinsing with PBS and mounting on slides with mounting medium containing DAPI, images were captured with the Biozero BZ-X700 digital microscope. The ratio of positive staining area to total area was calculated using ImageJ version 1.45 software.
To evaluate Nampt expression, we performed immunohistochemistry. After deparaffinization and antigen activation, the sections were incubated with a rabbit anti-Nampt monoclonal antibody (LS-C137911; LifeSpan BioSciences, Seattle, WA, USA) at 4 °C overnight. Subsequently, the sections were thoroughly rinsed with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ab6721; Abcam, Cambridge, UK) at room temperature for 1 h. After rinsing with PBS and mounting on slides with mounting medium containing DAPI, images were captured with the Biozero BZ-X700 digital microscope. The ratio of positive staining area to total area was calculated using ImageJ version 1.45 software.
6
Cardiac Fibrosis Quantification and Immune Cell Profiling
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The hearts were fixed with 4% paraformaldehyde, embedded in paraffin wax, sectioned into 3-μm thick slices, and stained with Masson’s trichrome. Fibrosis area was measured and analyzed using ImageJ analysis software (version 1.53; National Institutes of Health, Bethesda, MD, USA). For immunohistochemical examination, deparaffinization and antigen activation of the sections were performed, followed by incubation of the sections with rabbit polyclonal anti-NE antibodies (ab668672; Abcam, Cambridge, UK) or mouse anti-Ly6G antibodies (127,601; BioLegend, San Diego, CA, USA) at 4 °C overnight. Next, the sections were sufficiently rinsed in phosphate-buffered saline (PBS) and incubated with Alexa Fluor® 633-labeled goat antirabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) or Alexa Fluor® 546-labeled goat antimouse IgG (Thermo Fisher Scientific) at room temperature for 2 h. After rinsing in PBS and mounting onto slides using mounting medium with DAPI, fluorescent images of the slides were captured using a digital fluorescence microscope (Biozero BZ-X700; Keyence, Osaka, Japan).
7
Evaluating Pir-PLGA NP-Loaded AdSC Migration
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The migration activity of Pir-PLGA NP-loaded AdSCs was evaluated by a migration assay using a TranswellTM insert with a 5.0 μm pore size for 24-well plates (Sigma-Aldrich). Briefly, the Pir-PLGA NPs or PLGA NPs (0, 10, 25, 50 and 100 µg) were incorporated into the AdSCs (1.0 × 105 cells). Two different approaches were adopted. First, a plate well was filled with AdSC growth medium (DMEM/F12 600 μl) with 20% FBS. The insert chamber was used to accommodate 2.0 × 104 AdSCs in DMEM/F12 (200 μl) with 0% FBS. A second plate well was filled with tumor growth medium (DMEM 600 μl) with 10% FBS and 5.0 × 104 KP1N cells. AdSCs in DMEM/F12 (200 μl) with 10% FBS were placed in the insert chamber. The cells were allowed to migrate for 4 h or 16 h at 37 °C, immediately followed by cell fixation with 4% paraformaldehyde (PFA). The nuclei were counterstained with DAPI (Sigma). The number of AdSCs on the basolateral side was counted under a computer-assisted fluorescence/light microscope BioZero BZ-X700 (Keyence, Osaka, Japan).
8
Xenograft Tumor Characterization via Fluorescent Immunostaining
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The KP1N-derived xenografts were harvested on day 42. The tumors were fixed for 6 h in 4% PFA and incubated overnight in a 15% sucrose solution. The tissues were embedded in optimal cutting temperature (OCT) compound (Sakura FineTek, Japan) and sectioned at a 6-mm thickness. Fluorescent immunostaining was performed to detect tumor apoptosis and vascularity. The TUNEL assay (DeadEndTM Fluorometric TUNEL system, Promega) was used as a marker for apoptotic cells. Isolectin B4 (ILB4) (1:100; Vector Laboratories) was used for capillary staining with the DyLight 549 streptavidin-biotin binding method. Anti-mouse CD3 and F4/80 antigen (1:100; Thermo Fisher Scientific) were used for staining inflammatory cells with a secondary antibody of Alexa Fluor 594-conjugated IgG. The nuclei were counterstained with DAPI, and the sections were mounted in aqueous mounting medium. The images were examined under a computer-assisted fluorescence/light microscope (BioZero BZ-X700, Keyence, Osaka, Japan).
9
Immunocytochemical Analysis of hBMSCs under pH Stress
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For immunocytochemical analysis, hBMSCs were cultured under pH 7.4 or pH 6.8 for 48 h and then fixed with 4% PFA for 20 min, washed in PBS, blocked with 5% goat serum and then immunolabeled with primary antibody, or the isotype-matched IgG antibody. The target proteins were visualized with secondary antibody conjugated with Alexa Fluor 488 or 647 (Life Technologies) under a fluorescence microscope (Biozero BZ-X700, KEYENCE). Antibodies for Ki-67 was purchased from Abcam (Cambridge, UK).
10
Pir-PLGA NPs Enhance AdSCs Apoptosis
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Pir-PLGA NPs (0, 10, 25, 50 and 100 µg) were incorporated into the AdSCs (1.0 × 105 cells), and the Pir-PLGA NP-loaded AdSCs were cultured in DMEM/F12 containing 10% FBS for 48 h at 37 °C. The nuclei were counterstained with DAPI, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (In Situ Cell Detection Kit, TMR red, Roche) was performed, and the cells were immediately observed under a computer-assisted fluorescence/light microscope (BioZero BZ-X700, Keyence, Osaka, Japan). For the coculture assays, 50 µg of Pir-PLGA NPs was incorporated into the AdSCs (1.0 × 105 cells). The KP1N cells were cocultured with AdSCs using transwells. A total of 1.0 × 105 KP1N cells and 1.0 × 104 AdSCs at different ratios (10:1) were cultured in complete DMEM with 10% FBS for 48 h. The KP1N cell nuclei were counterstained with DAPI, a TUNEL assay was performed, and the cells were immediately observed under a computer-assisted fluorescence/light microscope (BioZero BZ-X700).
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