MDA-MB-231 cells were allowed to grow in 6-well plates supplemented with DMEM media to reach 70–80% confluency as described above. The cells were then incubated in reduced serum medium for 6 h containing 1% FBS. Cell cultures were scratched with a 200 µL sterile pipette tip and washed with PBS to remove detached cells and debris. Two crosses were scratched in each well, and the scratches were immediately subjected to photography using Nikon Inverted Microscope at 20 × magnification. Cells were then transfected with miR-941 inhibitor (50 nM) by using lipofectamine 3000. After 24 h, images of the same areas were acquired by using Nikon Inverted Microscope at 20 × magnification. The quantitative values of the percentage of cells covered in the scratch after 24 h were determined by using the web-based WimScratch module of Wimasis online software as described28 . At least three biological replicates per experiment were used and the presented results were representative of triplicate experiments with similar outcome.
Inverted microscope
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Canada, France, Italy, Netherlands, China
The Nikon Inverted Microscope is a laboratory instrument designed for the observation and analysis of samples. Its core function is to provide a magnified view of specimens from below, allowing for efficient examination of cell cultures, tissue samples, and other materials.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (52 mentions)
Crystal violet, by Merck Group (17 mentions)
Transwell chamber, by Corning (14 mentions)
Matrigel, by Corning (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Fura 2 am, by Thermo Fisher Scientific (9 mentions)
Crystal violet, by Beyotime (9 mentions)
Paraformaldehyde (pfa), by Merck Group (8 mentions)
Proscan 2, by Prior Scientific (7 mentions)
Nis elements software, by Nikon (7 mentions)
Matrigel, by Merck Group (7 mentions)
Image pro plus 6, by Media Cybernetics (6 mentions)
Ab6721, by Abcam (6 mentions)
Culture insert 2 well, by Ibidi (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Crystal violet, by Solarbio (5 mentions)
Image pro plus software, by Media Cybernetics (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Paraformaldehyde (pfa), by Solarbio (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
910 protocols using inverted microscope
1
Wound Healing Assay of MDA-MB-231 Cells
2
Cell Invasion and Wound-Healing Assay
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For the invasion assay, the cells (1×104 cells/well) incubated in serum-free medium were added to the upper chamber of the insert with Matrigel (BD Biosciences, San Jose, CA, USA). Following incubation at 37°C for 24 h, permeable cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 10 min at room temperature. The cells were counted under an inverted microscope (Nikon Corp.) in 5 different visual fields and averaged.
For the wound-healing assay, the cells (5×104 cells/well) were seeded into 6-well plates and incubated in serum-free medium for 24 h at 37°C. The cell monolayer was scratched with a 200-µl pipette tip to form wound gaps. The cells were then washed in PBS and then incubated for 24 h at 37°C continuously, and images were captured under an inverted microscope (Nikon Corp.) at the indicated time-points.
For the wound-healing assay, the cells (5×104 cells/well) were seeded into 6-well plates and incubated in serum-free medium for 24 h at 37°C. The cell monolayer was scratched with a 200-µl pipette tip to form wound gaps. The cells were then washed in PBS and then incubated for 24 h at 37°C continuously, and images were captured under an inverted microscope (Nikon Corp.) at the indicated time-points.
3
Osteogenic Differentiation of Endothelial Progenitor Cells
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The EPCs were treated with TBHP (2 h), and then cells were cultured in routine DMEM medium (without TBHP) for 6 days. After that, the EPCs were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min and with alizarin red solution (Beyotime, Shanghai, China) for 30 min at 37°C. The stained cells were observed, and images were captured with an inverted microscope (Nikon, Tokyo, Japan). A BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China) was utilized based upon provided directions. Briefly, cells were washed three times by using PBS and fixed with 4% paraformaldehyde for 15 min. BCIP/NBT substrate was then used to treat cells for 24 h, the stained cells were observed, and images were captured with an inverted microscope (Nikon, Tokyo, Japan).
4
Evaluating XGC-1 Cell Migration
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The migration of XGC-1 cells was analyzed by wound healing and transwell assays. For wound healing assay, a wound on the cell layer was created using a pipette tip (100 μL) when the transfected XGC-1 cells reached 90% con uence. The pictures of the cells at 0 h and 24 h were photographed with an inverted microscope (Nikon). The migration rate was calculated according to the following equation: cell migration rate (%) = (1-the distance following healing/the distance prior to healing) ×100%.
For transwell migration assay, the serum-free containing transfected XGC-1 cells (1×10 5 cells) was added to the top chamber of the transwell chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was supplemented into the bottom of the transwell chamber. After removing the cells on the upper surface of the membrane, the remaining cells were xed and stained with paraformaldehyde (4%, Sigma) and crystal violet (0.5%, Sigma), respectively. The migrating cells were calculated with an inverted microscope (Nikon) at 100 × magni cation.
For transwell invasion assay, its method was the same as the cell migration assay. It was worth noting that the transwell chamber of the invasion assay was pre-coated with Matrigel (Sigma).
For transwell migration assay, the serum-free containing transfected XGC-1 cells (1×10 5 cells) was added to the top chamber of the transwell chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was supplemented into the bottom of the transwell chamber. After removing the cells on the upper surface of the membrane, the remaining cells were xed and stained with paraformaldehyde (4%, Sigma) and crystal violet (0.5%, Sigma), respectively. The migrating cells were calculated with an inverted microscope (Nikon) at 100 × magni cation.
For transwell invasion assay, its method was the same as the cell migration assay. It was worth noting that the transwell chamber of the invasion assay was pre-coated with Matrigel (Sigma).
5
Quantifying Adiposome-like Droplets in A549 Cells
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The adiposome-like droplets were frequently observed in nearly all of the A549 cancer cells at 1 week after 50 μM PGZ treatment. Neutral triglycerides and lipids in the adiposome-like vesicles were stained with Oil Red O solution. Briefly, the cells were washed in D-PBS and treated with a fixation solution overnight. And the cells were stained with 0.5% Oil Red O solution for 2 h. The lipid droplets stained with red color were investigated under an inverted microscope (Nikon, Japan). Further, the cell volume was calculated in the A549 cancer cells treated with 50 μM PGZ for 4 weeks. Every week, the cell diameter of over 100 cells was evaluated under an inverted microscope (Nikon, Japan) equipped with a CCD camera and image software (Nikon, Japan), followed by cell harvesting using 0.25% trypsin EDTA solution. The cell volume was calculated from cell diameter.
6
Saponin Effects on Follicular Maturation
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IVM process was performed on the basis of Rajabi-Toustani and coworkers protocol with some modifications (7 ).
Maturation medium consisted of α-MEM supplemented with, 5% FBS, 7.5 IU/ml recombinant human rhFSH (Organon, Holand), 100 IU/ml HCG (Organon, Holand) and 5 ng/ml ITS (Gibco, USA). The preantral follicles were cultured in α-MEM supplemented with different concentrations of sea cucumber saponin (0, 1, 2, 4, and 8 μg/ml) and were covered with mineral oil for 12 days. Then, the effect of sea cucumber saponin on follicular growth and maturation was analyzed. Follicular viability was evaluated by Trypan blue staining. At various intervals from the onset of incubation, oocytes were observed by inverted microscope (Nikon, Japan), and nucleus morphological changes of GV and germinal vesicle break down were evaluated, or the extrusion of first polar body (Metaphase II: MII) using inverted microscope observed quantitatively.
Maturation medium consisted of α-MEM supplemented with, 5% FBS, 7.5 IU/ml recombinant human rhFSH (Organon, Holand), 100 IU/ml HCG (Organon, Holand) and 5 ng/ml ITS (Gibco, USA). The preantral follicles were cultured in α-MEM supplemented with different concentrations of sea cucumber saponin (0, 1, 2, 4, and 8 μg/ml) and were covered with mineral oil for 12 days. Then, the effect of sea cucumber saponin on follicular growth and maturation was analyzed. Follicular viability was evaluated by Trypan blue staining. At various intervals from the onset of incubation, oocytes were observed by inverted microscope (Nikon, Japan), and nucleus morphological changes of GV and germinal vesicle break down were evaluated, or the extrusion of first polar body (Metaphase II: MII) using inverted microscope observed quantitatively.
7
Evaluating XGC-1 Cell Migration
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The migration of XGC-1 cells was analyzed by wound healing and transwell assays. For wound healing assay, a wound on the cell layer was created using a pipette tip (100 μL) when the transfected XGC-1 cells reached 90% con uence. The pictures of the cells at 0 h and 24 h were photographed with an inverted microscope (Nikon). The migration rate was calculated according to the following equation: cell migration rate (%) = (1-the distance following healing/the distance prior to healing) ×100%.
For transwell migration assay, the serum-free containing transfected XGC-1 cells (1×10 5 cells) was added to the top chamber of the transwell chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was supplemented into the bottom of the transwell chamber. After removing the cells on the upper surface of the membrane, the remaining cells were xed and stained with paraformaldehyde (4%, Sigma) and crystal violet (0.5%, Sigma), respectively. The migrating cells were calculated with an inverted microscope (Nikon) at 100 × magni cation.
For transwell invasion assay, its method was the same as the cell migration assay. It was worth noting that the transwell chamber of the invasion assay was pre-coated with Matrigel (Sigma).
For transwell migration assay, the serum-free containing transfected XGC-1 cells (1×10 5 cells) was added to the top chamber of the transwell chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was supplemented into the bottom of the transwell chamber. After removing the cells on the upper surface of the membrane, the remaining cells were xed and stained with paraformaldehyde (4%, Sigma) and crystal violet (0.5%, Sigma), respectively. The migrating cells were calculated with an inverted microscope (Nikon) at 100 × magni cation.
For transwell invasion assay, its method was the same as the cell migration assay. It was worth noting that the transwell chamber of the invasion assay was pre-coated with Matrigel (Sigma).
8
Wound Healing and Cell Migration Assays
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The monolayer cells were scraped by the tip of 200-μL pipette, and then cultured in the medium with 2% FBS. The wound healing was observed under the Inverted microscope (Nikon, Japan) and photos were taken at 0 hr and 24 hrs.
Transwell, the upper chamber was added with Matrigel in advance, and then 200 μL 1×105/mL transfected cells with 2% FBS was seeded and incubated on the upper chamber, the bottom chamber was added 600 μL medium with 20% FBS. After 48 hrs, remove the cells and Matrigel in the upper chamber and fix the bottom chamber of migration cells with formaldehyde. The number of cells were measured by Inverted microscope (Nikon, Japan)
Transwell, the upper chamber was added with Matrigel in advance, and then 200 μL 1×105/mL transfected cells with 2% FBS was seeded and incubated on the upper chamber, the bottom chamber was added 600 μL medium with 20% FBS. After 48 hrs, remove the cells and Matrigel in the upper chamber and fix the bottom chamber of migration cells with formaldehyde. The number of cells were measured by Inverted microscope (Nikon, Japan)
9
Transwell Assay and Scratch Wound Healing
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Transwell assay: The transwell co-culture system was employed to evaluate the effect of PRP-Exos on TSPCs migration. A total of 5 × 104 cells/well were uniformly seeded in the upper chamber of transwell plates (8-μm pore size, six-well format, Corning, USA), and 500 μL of serum-free medium was added. In the lower chamber, 600 μL of PRP-Exos medium (supplemented with 10% FBS, Thermo Scientific) at varying concentrations (0, 20, and 50 μg/mL) was introduced. Following 24 h of incubation, non-migrated cells in the upper layer of the upper chamber were gently wiped off, and the migrated cells on the permeable membrane were fixed with 4% paraformaldehyde (Biorigin) and subsequently stained with crystal violet. Using an inverted microscope (Nikon, Japan), we examined five randomly selected fields of view and counted the number of migrated cells.
Scratch wound-healing assay: TSPCs (5 × 104 cells) were uniformly seeded in six-well plates. When the cells' confluency achieved 80–90% confluency, a straight and consistent scratch across the cell monolayer was created using a P200 pipette tip. Subsequently, cell debris was removed by rinsing with PBS, and 2 mL of serum-free medium containing varying concentrations of PRP-Exos was introduced into each well. Cell migration was documented at 0 and 24 h for all cell groups using an inverted microscope (Nikon).
Scratch wound-healing assay: TSPCs (5 × 104 cells) were uniformly seeded in six-well plates. When the cells' confluency achieved 80–90% confluency, a straight and consistent scratch across the cell monolayer was created using a P200 pipette tip. Subsequently, cell debris was removed by rinsing with PBS, and 2 mL of serum-free medium containing varying concentrations of PRP-Exos was introduced into each well. Cell migration was documented at 0 and 24 h for all cell groups using an inverted microscope (Nikon).
10
Analyzing Cell Migration and Invasion
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The migration of XGC-1 cells was analyzed by wound-healing and transwell assays. For the wound-healing assay, wounds on cell layer were created using a pipette tip (100 μL) when the cells reached 90% confluence. Then, FBS-free medium was used to rinse the created wound to remove all exfoliated cell debris. Photographs were taken at 0 h and 24 h with an inverted microscope (Nikon). Percentage of migration was calculated according to the following equation: cell migration rate (%) = (1-the distance following healing/the distance prior to healing) × 100%.
For the transwell migration assay, serum-free medium containing transfected XGC-1 cells (1×105 cells) was added to the top chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was added to the bottom chamber. After removing the cells on the upper surface of the membrane, the remaining cells were fixed with paraformaldehyde (4%, Sigma) and stained with crystal violet (0.5%, Sigma). The number of migrating cells was calculated with an inverted microscope (Nikon) at 100 × magnification.
The procedure of the transwell invasion assay was the same as the transwell migration assay. It was worth noting that the transwell chamber used in the invasion assay had been pre-coated with Matrigel (Sigma).
For the transwell migration assay, serum-free medium containing transfected XGC-1 cells (1×105 cells) was added to the top chamber (8 μm, Costar, Cambridge, MA, USA). The cell medium containing 10% FBS was added to the bottom chamber. After removing the cells on the upper surface of the membrane, the remaining cells were fixed with paraformaldehyde (4%, Sigma) and stained with crystal violet (0.5%, Sigma). The number of migrating cells was calculated with an inverted microscope (Nikon) at 100 × magnification.
The procedure of the transwell invasion assay was the same as the transwell migration assay. It was worth noting that the transwell chamber used in the invasion assay had been pre-coated with Matrigel (Sigma).
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