Cells were seeded (1x105 cells/well) into 12-well plates. At 80% confluence, the medium was replaced with serum-free DMEM and cells were incubated at 37˚C overnight. Subsequently, a 200-µl pipette tip was used to scratch the cell monolayer. Following washing with PBS to remove free-floating cells and debris, the plates were maintained at 37˚C with 5% CO2. Following incubation for 48 h, the wounds were observed using a BX51 inverted microscope (Olympus Corporation; magnification, x100). Cell migration was quantified as follows: (0 h scratch width-scratch width following culturing)/0 h scratch width.
Bx51 inverted microscope
Manufactured by Olympus
Sourced in United States, Japan
The BX51 inverted microscope is a high-performance optical instrument designed for a variety of laboratory applications. It features a sturdy, ergonomic design and advanced optical components that deliver clear, detailed images. The BX51 is capable of various observation techniques, including brightfield, darkfield, and phase contrast, making it suitable for a wide range of sample types and research projects.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Fitc conjugated anti rabies monoclonal antibody, by Fujirebio (2 mentions)
Bx51 microscope, by Olympus (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ab4448, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ab28144, by Abcam (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
23 protocols using bx51 inverted microscope
1
Scratch Wound Healing Assay
2
Histological Analysis of Knee Joint Tissues
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All the knee joint tissues were stained with H&E following the manufacturer’s standard protocol (Newcomer Supply, Middleton, WI, USA). Stained slides were analyzed for the presence or absence of inflammation and fatty infiltration. All the slides were reviewed by a board-certified pathologist. All the images were scanned at × 20 magnification using an Olympus BX51inverted microscope (Olympus Scientific Solutions, Waltham, MA, USA) with a scale bar of 200 μm. The average adipocyte size in infra- and suprapatellar fat was calculated by selecting 15 random adipocytes in the different swine groups in each category using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA). Chondrocyte counting was done using three high-power field images, and the average number of clustered chondrocytes to total chondrocytes per high-power field in each group was calculated. Areas of fatty infiltration were scanned, and fatty infiltration within the matrix of muscle, tendon, and ligament was calculated by measuring the area of all adipocytes present in the matrix followed by calculating the percentage of infiltration area per field.
3
Quantitative Collagen Staining Analysis
4
Matrigel and Wound Healing Assays for Cell Invasion and Migration
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Matrigel and the wound healing assay were used to detect cell invasion and migration, respectively. For cell invasion, 100 µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA; serum-free medium diluted 1:6) was added to the upper chamber of a 24-well plate Transwell chamber and then placed in a 37°C 5% CO2 incubator for 4-6 h to form a gel. Subsequently, 100 µl transfected cell suspension (1×105) was added in the upper chamber, while 500 µl complete medium was added into the bottom chamber. Following overnight culture so that the cells invade to the lower surface of the filter, cells were washed and fixed with 4% paraformaldehyde for 30 min. Finally, invaded cells were stained with 0.1% crystal violet for 20 min and 5 visual fields were selected randomly using a BX51 inverted microscope (Olympus Corporation, Tokyo, Japan) at magnification, ×100.
For the wound healing assay, cells were cultured in RPMI-1640 medium in six-well plates (5×105 cells/ml) for 24 h, then a wound was made in each plate with a 100 µl plastic pipette tip. After being washed three times with PBS, the cells were cultured for another 24 h. The wound width was then observed in each well using an Olympus BX51 microscope (Olympus Corporation) at magnification, ×200 in five random fields. Each sample was performed in triplicate.
For the wound healing assay, cells were cultured in RPMI-1640 medium in six-well plates (5×105 cells/ml) for 24 h, then a wound was made in each plate with a 100 µl plastic pipette tip. After being washed three times with PBS, the cells were cultured for another 24 h. The wound width was then observed in each well using an Olympus BX51 microscope (Olympus Corporation) at magnification, ×200 in five random fields. Each sample was performed in triplicate.
5
Quantitative Analysis of Neuronal and Astrocytic Gene Expression
6
Immunofluorescence Localization of InvX Proteins
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E. coli (UT4400/pMKinvX) cells were fixed onto microscope slides with 0.4% paraformaldehyde for 10 min at room temperature, and non-specific binding was blocked by incubation in 1% (wt/vol) BSA for 30 min. Slides were incubated for 1 h with a 1:200 dilution of a rabbit Ab raised against InvXa, InvXb, InvXc, and InvXd, washed, and incubated with 1:1000 dilution of fluorescein isothiocyanate-labeled anti-rabbit Ab (Abcam Plc, Cambridge, UK) for 30 min. Normal rabbit IgG was used for labelling for the negative control. After extensive washing, the coverslips were mounted. Slides were viewed on an Olympus BX51 inverted microscope with an epifluorescence attachment.
7
Fixing and Staining A2780 and SKOV3 Cells
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The 3 groups of A2780 and SKOV3 cells were fixed using methanol acetic acid for 15 min and then stained with Hoechst 33342 for 5 min. Being washed twice with PBS, the cells were immediately photographed under an Olympus BX51 inverted microscope at ×400 magnification (Olympus Corp., Tokyo, Japan).
8
Scratch Assay for Cell Migration
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Untransfected and transfected cells were seeded at 5.0×105 cells/well in 6-well plates and cultured routinely. After reaching 90% confluence, the cell monolayer was scratched with a sterile pipette tip. After washing 3 times with PBS for 5 min each to clear the floating cells, 1.5 ml MEM/F12 and RPMI-1640 medium supplemented with 1% FBS were added into each well. Photographs were taken by an Olympus BX51 inverted microscope at ×100 magnification (Olympus Corp.) at 0, 24 and 48 h after scratching. Results were indicated as the relative width of scratch-the distance migrated relative to the original scratched distance. The experiment was conducted 3 times.
9
Colony Formation Assay with OLA
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Cells were plated in growth medium (GM) in 6-multiwell plates. The next day, they were treated with vehicle (−) or OLA at 50 and 100 μM. OLA treatment was renewed every 3 days. After 14 days, surviving colonies were stained with Crystal Violet and the images were acquired by Olympus BX51 inverted microscope. The obtained data by the Image J software were elaborated and normalized.
10
Visualizing Centrosome Components in GFP-APC Cells
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U2OS cells were plated onto coverslips in 6-well plates and grown until 60%–70% confluency before transfection of GFP-tagged APC fragment sequences for 48 h (see Section 4.2 ). Cells were fixed with methanol:acetone (1:1) for 3 min, followed by three washes using PBS. Cells were blocked with 3% bovine serum albumin and stained with primary antibodies. To detect the centrosome, polyclonal PCNT (Abcam ab4448, 1:1500) and PCNT monoclonal (Abcam ab28144, 1:1500) were used. γ-tubulin (Sigma T5192, 1:800) and monoclonal γ-tubulin (Sigma T5326, 1:800) antibodies have also been used to yield a similar localization result. Cells were washed three times using PBS and subsequently incubated with the fluorescence secondary probes Alexafluor-594 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed extensively before they were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). Cells were stained in the dark to prevent bleaching of GFP. Cells were visualized using Olympus BX-51 inverted microscope (Olympus) and optical sections were taken using an Olympus FV1000 confocal microscope (Olympus) at 60× magnification.
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