The adherent cells were scraped vertically using the 200-μL pipette and cultured with the medium containing 1% FBS. The wound healing was confirmed under an inverted microscope (Zeiss, Japan) at 0 and 24 hours, and photos were taken. In the transwell assay, total 1 × 104 transfected cells were added and cultured on the upper chamber with Matrigel (BD Bioscience, CA, USA) coated, and the bottom chamber was seeded 500 μL medium with 10% FBS. After 48 h incubation, the upper chamber was removed and the bottom chamber of cells was fixed using formaldehyde. The invaded cell number was counted via an inverted microscope (Zeiss, Japan).
Inverted microscope
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom, Japan, China, Switzerland, Australia
The Inverted microscope by Zeiss is a specialized optical instrument designed for observing samples from the underside. It allows for the examination of cells, tissues, and other specimens in their natural state, making it particularly useful for cell culture and live-cell imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (24 mentions)
Axiovision software, by Zeiss (10 mentions)
Matrigel, by Corning (8 mentions)
Triton x 100, by Merck Group (6 mentions)
Transwell chamber, by Corning (6 mentions)
Toluidine blue, by Merck Group (5 mentions)
Nucblue fixed cell stain readyprobe, by Thermo Fisher Scientific (5 mentions)
Apotome, by Zeiss (5 mentions)
Transwell insert, by Corning (4 mentions)
Dexamethasone (dex), by Merck Group (4 mentions)
Oil red o, by Merck Group (4 mentions)
Fluorescence microscope, by Zeiss (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Triton x 100, by MP Biomedicals (3 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (3 mentions)
6 well plate, by Corning (3 mentions)
Quantifier, by Optronics (3 mentions)
Elisa reader, by Molecular Devices (2 mentions)
490 protocols using inverted microscope
1
Cell Migration and Invasion Assay
2
Cell Viability Assays for SCRT
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Cell growth was assessed using the trypan blue dye exclusion assay. In brief, cells (2 × 104 cells/well) were seeded in 6-well plates. After treatment with the indicated concentrations of the SCRT for the indicated times, the cells were trypsinized and viable cells were counted by trypan blue dye exclusion using a hemocytometer under an inverted microscope (Carl Zeiss, Jena, Germany). Cell viability was determined using the MTT assay. In brief, cells (2 × 104 cells/well) were seeded in 24-well plates and exposed to the extracts of SCRT for the indicated times. After treatment, 5 mg/ml MTT solution was added, followed by 3 h incubation at 37°C in the dark, and the media was then removed. The formazan precipitate was dissolved in dimethyl sulfoxide, and absorbance of the formazan product was measured at a wavelength of 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA). For the morphological study, cells were photographed directly using an inverted microscope (Carl Zeiss, Oberkochen, Germany).
3
Quantifying Osteoclast Formation and Bone Resorption
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After culture with M-CSF and RANKL, mature osteoclasts were treated in 4% paraformaldehyde and stained with an Acid Phosphatase Leukocyte (TRAP) Kit (Sigma-Aldrich, USA), in accordance with the manufacturer's protocol. Using an inverted microscope (Zeiss, Germany), the numbers of TRAP-positive multinucleated cells with ≥3 nuclei were then counted to determine the number of osteoclast-like cells. To observe the bone resorption activity of mature osteoclasts, BMMs were cultured with M-CSF and RANKL in a 24-well osteo assay surface multiple-well plate (Corning Life Sciences, USA) coated with a thin inorganic three-dimensional crystalline material. After 6-8 days of culture, a pit formation assay was performed, and 100 μL of 10% bleach solution was added to each well. Cells were then incubated in the bleach solution for 10 minutes at room temperature. The wells were rinsed twice with distilled water and allowed to air dry at room temperature for 2 hours. Using an inverted microscope (Zeiss), analyses of the individual pits or multiple pit clusters were performed at 5× magnification.
4
Evaluating Bioprinted ARPE-19 Cell Viability
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The cell viabilities of bioprinted ARPE-19 cells at day 1, 7 and 14 were evaluated by prestoblue (Thermo Fisher, Grand Island, NY, USA) assay in test media (1 mL) consisted of prestoblue (10%) and FBS (5%). Control groups were cells without bioprinting. Then, the cell viabilities were calculated according to vendor’s protocol. Meanwhile the bioprinted ARPE-19 cells at day 1, 7 and 14 were fixed in 4% paraformaldehyde; then, they were stained by ActinGreen™ 488 ReadyProbes® and NucBlue® Live ReadyProbes® reagents and observed under inverted microscope (Zeiss). The bioprinted ARPE-19 cells on ultrathin membrane at day 14 were also observed under inverted microscope, and the sample was then fixed by 4% paraformaldehyde and HE stained. The bioprinted samples were stained by ZO-1 Monoclonal Antibody, FITC (ZO1-1A12)/NucBlue® Live ReadyProbes® reagents and ActinGreen™ 488 ReadyProbes®/ NucBlue® Live ReadyProbes® reagents and live/dead assay, respectively. The stained samples were observed under inverted microscope (Zeiss) and laser scanning microscope (Zeiss LSM 710). For scanning electron microscope (SEM, JEOL) observation, the samples were fixed at day 1, 7 and 14, and then were dehydrated in 30%, 50%, 75%, 95% and 100% ethanol gradually before critical-point drying, and the samples were sputter-coated (sputtering time 90 s and current 20 mA) by gold before SEM observation.
5
Evaluating Cell Migration and Invasion
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To evaluate cell migration, wound lines in the form of a cross were made by scraping with a plastic 200‐μL pipette tip in confluent cells. After wounding, floating cells were washed out with PBS and were incubated with 1% FBS‐containing medium supplemented with or without EECU for 24 hr. Subsequently, the width of wound healing was photographed under an inverted microscope (Carl Zeiss). In vitro invasive activity was assessed using the Trans‐well chamber system (10 mm diameter, 8 μm pore size with polycarbonate membrane; Corning Costar Corp., Cambridge, MA, USA). B16F10 cells were kept in serum‐free medium for 24 hr. The cells (5 × 104 cells/well) were placed in the upper chamber of trans‐well insert, and at the same time, 10% FBS‐containing complement medium supplemented with EECU of 0, 20, and 40 μg/mL was added into the lower chamber, and then cells were incubated for 24 hr. Cells that invaded through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E; Sigma‐Aldrich Chemical Co.). The stained cells were counted and observed under an inverted microscope (Carl Zeiss).
6
Cell Migration Assay Using Transwell
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Cells of each experimental group were resuspended and seeded in upper transwell chambers with pore size of 8 µm for the migration assay (Corning, NY, United States) at a density of 1 × 106 cells/ml. The cells were allowed to cross the chamber for 48 h. The penetrated cells were fixed, stained with 1% crystal violet, and counted under an inverted microscope at a magnification ×100 (Zeiss, Thornwood, United States).
7
Quantifying Cellular Proliferation via EdU
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EdU Cell Proliferation Kit (Beyotime, C0071) was used to detected GC proliferation according to the manufacturer’s instructions. The images were blindly captured by using a Zeiss inverted microscope and three microscopic fields of each slide were quantified for each group with Image J.
8
Immunocytochemistry of Induced Pluripotent Stem Cells
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Immunocytochemistry was performed as previously described (22 (link)). Fixed iPSCs with 4% formaldehyde were permeabilized with 0.1% Triton X-100 in 0.1% PBS/Tween for 30 min at room temperature and blocked using a blocking solution consisting of 0.1% PBS/Tween with 3% BSA for 30 min at room temperature. After blocking, the cells were incubated with primary antibody (TRA-1-60, SSEA4, and OCT4, Stemgent) solution diluted with 0.1% PBS/Tween (1:100) in 4°C overnight. Cells were washed three times with 0.1% PBS/Tween and incubated for 1 hour at room temperature with a secondary antibody solution (1:1,000). Samples were imaged and captured using a Carl Zeiss inverted microscope.
9
Exploring BV-CNP Effects on Yeast-Hyphal Transition
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Spider medium (1% nutrient broth, 1% mannitol, 0.2% K2HPO4, pH 7.2) was used to explore the effects of BV-CNPs on the yeast-to-hyphal morphological transition according to Sharaf (2020 (link)). The cell suspension of UCF was adjusted at a concentration of 1 × 106 CFU/ml in spider medium and transferred 200 µl into each well of 96-well plates. Then UCF were treated with different concentrations (one quarter, one half, and entire MIC value) of BV-CNPs corresponding to each strain in addition to control (the treatment was replaced with distilled water). After incubation at 37 °C for 8 h, the morphologies of cells exposed to different concentrations of BV-CNPs were examined under Zeiss inverted microscope (Germany).
10
Wound Healing Assay with huc-MSCs
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To conduct the wound healing assay, huc-MSCs in the logarithmic growth phase were inoculated in 6-well plates (3 × 105/well) and artificially scratched in cross form, followed by treatment of PL with serum-free medium. The cells were observed and imaged at four different time points (0, 12, 24, and 36 h) under an inverted microscope (CarlZeiss, Göttingen, Germany). The wound area was calculated with Image J 1.47 software. Each experiment was conducted in triplicate.
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