Scratch assay and transwell assay to detect the migrated ability. Scratch assay was performed by a 200 μl pipette in 60 mm dishes when cells grew up to 90–100% density. And the floated cells were removed with twice Hank's (Beyotime, C0218) washes, and wound pictures were taken at 0 hour, 24 hours and 48 hours by Olympus IX70 inverted microscope. In transwell assay, bottom chamber was filled with 1 ml RPMI-1640 with 10% fetal bovine serum and 1% penicillin/streptomycin and the top chamber was filled with the same media with 4 × 104 (PC3 cells with ectopic expression of UBE2T) or 2 × 104 (Du145 cells with ectopic expression of UBE2T) or 4 × 104 (LNcaP cells with ectopic expression of UBE2T) cells suspended. The cells were incubated for 48 hours and the fixed in methanol longer than 30 minutes and stained with Giemsa stain after the cells on the top surface of membrane removed by cotton swab. The pictures were taken by Olympus IX70 inverted microscope and the numbers of migrated cells were counted.
Ix70 inverted microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, United Kingdom
The Olympus IX70 is an inverted microscope designed for biological and life science research applications. It features a stable and ergonomic design, supporting a range of imaging techniques and accessories to facilitate various microscopy workflows.
Automatically generated - may contain errors
Lab products found in correlation
86000 sedat quadruple filter set, by Chroma Technology (5 mentions)
Deltavision core system, by Cytiva (5 mentions)
Matrigel, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Dual excitation fluorescence photomultiplier system, by IonOptix (4 mentions)
Confocal laser scanning microscopy, by Leica (4 mentions)
Fluoview 500, by Olympus (4 mentions)
Rnaimax, by Thermo Fisher Scientific (4 mentions)
Iq software, by Oxford Instruments (4 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Fluoview fvx confocal scan head, by Olympus (3 mentions)
24 well tissue culture plate, by BD (3 mentions)
Normal goat serum (ngs), by Zhongshan Golden Bridge Biotechnology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (3 mentions)
Microfire, by Olympus (3 mentions)
Incubator box, by Olympus (3 mentions)
Csu10 scanhead, by Olympus (3 mentions)
296 protocols using ix70 inverted microscope
1
Evaluating Cell Migration Capacity
2
Assessment of Cardiomyocyte Mechanics and Calcium Signaling
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Mechanical properties of cardiomyocytes were assessed using a Softedge MyoCam system (IonOptix Corporation, Milton, MA, USA) equipped with an IX-70 Olympus inverted microscope. Cardiomyocytes were electrically stimulated at 0.5 Hz in a contractile buffer containing NaCl 135 mM, KCl 4.0 mM, CaCl2 1.0 mM, MgCl 1.0 mM, glucose 10 mM and HEPES 10 mM. Cell shortening was assessed including peak shortening (PS), maximal velocity of shortening (+dL/dt), maximal velocity of re-lengthening (−dL/dt), time-to-PS (TPS), and time-to-90% re-lengthening (TR90). For intracellular Ca2+ recording, cardiomyocytes were loaded with Fura-2/AM (0.5 μM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). To assess intracellular Ca2+ signaling, cells were exposed to light emitted by a 75-W lamp and passed through 360 nm or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm and the alterations in fura-2 fluorescence intensity (FFI) were quantitated from the FFI ratio at 360 nm to 380 nm. Fluorescence decay time was assessed as an indicator of intracellular Ca2+ clearing [57 (link), 61 (link)].
3
Quantifying Myotube Formation in Muscle Cells
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Cell cultures were visualized with an IX70 Olympus inverted microscope under the 20X objective (yielding a total magnification of 200X), and representative phase contrast and DAPI images were captured as high-resolution JPG files with an Olympus camera with Magnafire digital imaging software. Composite images were created through GNU Image Manipulation Program (GIMP 2) which was used to identify myoblasts (defined as cells with one or two nuclei) or myotubes (defined as cells with three or more nuclei). DAPI staining was used to observe and count nuclei. These images were utilized to quantify myotube formation by modifying a myoblast fusion index paradigm [33 (link)] into a myotube formation index used in our laboratory [26 (link), 28 (link), 29 (link)]. Six composite images were analyzed for untreated cultures, and five composite images each for cultures exposed to 1 μg/mL NaNO3 or 10 μg/mL NaNO3 for the last 48 h of 72 h in DM. The myotube formation index was then calculated as the fraction of nuclei in myotubes, with the data displayed in Table 1 .
Myotube formation index
| Total nuclei | Nuclei in myotubes | Nuclei in myoblasts | Fraction of nuclei in myotubes | |
|---|---|---|---|---|
| Untreated (n = 6) | 2440 | 2177 | 263 | 0.8922 |
| 1 μg/ml NaNO3 (n = 5) | 2372 | 2088 | 284 | 0.8803 |
| 10 μg/ml NaNO3 (n = 5) | 1917 | 1653 | 264 | 0.8623 |
4
Quantifying Cell Migration Using Wound Healing Assay
5
Liposome Microscopy Visualization
6
Cardiomyocyte Contractility and Calcium Dynamics
7
High-throughput 2D and 3D Hepatocyte Assay
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HepG2 cells were seeded using a Multiflo FX multi-mode automated reagent dispenser (Biotek, UK) at 2.0 × 10 5 cells per well (2D) in Ibidi 96 well black microplates (Ibidi, USA) and 200 cells per well (3D) in 96well ULA round-bottomed plates (Corning, UK). Cells were incubated for 24 h (2D) and 7 days (3D) at 37 °C, 5% CO 2 . 2D and 3D cell cultures were treated with 30 mM of APAP for 24 h. Cells were subsequently stained with Calcein AM (1.5μM) and Ethidium homodimer-1 (10μM) and cell death and viability data were measured using Operetta CLS High Content Analysis System (PerkinElmer, UK).
For cells assessed on the Mera system, HepG2 cells were cultured in ULA microplates for 7 days and transferred to Mera for treatment and staining. 3D microtissues were stained with Calcein AM (3μM) and Ethidium homodimer-1 (10μM) on Mera and were then transferred to a ULA microplate for imaging consistency. Pixel intensity was measured using an IX70 Olympus inverted microscope (Olympus, Tokyo, Japan).
For cells assessed on the Mera system, HepG2 cells were cultured in ULA microplates for 7 days and transferred to Mera for treatment and staining. 3D microtissues were stained with Calcein AM (3μM) and Ethidium homodimer-1 (10μM) on Mera and were then transferred to a ULA microplate for imaging consistency. Pixel intensity was measured using an IX70 Olympus inverted microscope (Olympus, Tokyo, Japan).
8
Quantifying Lipid Accumulation in Cells
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To confirm the lipid accumulation in cultured cells, Oil red O staining (Sigma-Aldrich; Merck KGaA) was performed. Eight days after induction, mature adipocytes were fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature and then stained with Oil red O working solution (6:4 of oil red stock solution: Distilled water) for 15 min. PBS was washed three times to remove the excess Oil red O dye. The images were observed under a parallel phase contrast microscope (Olympus IX70 inverted microscope, Olympus Optical CO, Ltd.). For quantitative analysis, the percentage of positively stained areas were calculated using imageJ. Results are expressed as percentage of Oil red O-stained area compared to control. For each sample, the experiments were performed in triplicates.
9
Osteoclast Differentiation Assay
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BMMs (5 x 104 cells/well) were seeded into a 96-well plate and cultured in α-MEM with 10% FBS, 30 ng/ml M-CSF and the indicated concentrations of 15d-PGJ2 in the absence or presence of 100 ng/ml RANKL. Cells were cultured for 5 days, and fresh media containing the appropriate chemicals was replaced every other day. Cells were fixed using 3.7% (v/v) formaldehyde and stained for tartrate-resistant acid phosphatase (TRAP) activity for 10 min at 37°C using the Acid Phosphatase Leukocyte kit (Sigma-Aldrich) as described previously [6 (link),26 (link)]. TRAP-positive multinucleated cells (MNCs) with more than three nuclei were quantified as differentiated osteoclasts using an Olympus IX70 inverted microscope (Olympus Optical, Tokyo, Japan) (100x magnification).
10
Immunofluorescence Staining of Neuronal Markers
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Immunofluorescence staining was performed using the method described in our previous publication4 (link),24 (link). The primary antibodies were: rabbit anti-GHRH (1:100, catalog No: ab187512, Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:50, catalog number BM0055; Boster Bioengineering, Wuhan, China), mouse anti-Gephyrin (1:50, Abcam, Cambridge, MA, USA), guinea pig anti-microtubule-associated protein 2 (MAP2) (1:200, Sysy, Goettingen, Germany), mouse anti-glutamate decarboxylase 67 (GAD67) (1:100, Abcam, Cambridge, MA, USA), and guinea pig anti-vesicular GABA transporter (VGAT) (1:200, Sysy, Goettingen, Germany). The secondary antibodies were: an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:50, Zhongshan Golden Bridge, Inc., Beijing, China), Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:200, Zhongshan Golden BridgeInc., Beijing, China), and Alexa Fluor 633-conjugated goat anti-guinea pig IgG antibody (1:50, Abcam, Cambridge, MA, USA). Finally, the samples were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma, St. Louis, MO, USA) for 5 min to identify the nuclei. Immunofluorescently labeled sections were examined with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and an Olympus IX 70 inverted microscope (Olympus America, Melville, NY, USA).
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