Breast cancer cell migration was assessed under exposure to CM2D/CM3D (10× concentrated) and/or Dox (100 nM) through an in vitro scratch assay, performed in accordance with [16 (link),18 (link)]. Briefly, 2 × 105 MDA-MB-231 cells were seeded in 24-well plates in complete culture medium. After 24 h, a scratch was performed using a 200 µL sterile pipette tip, and cells were incubated in serum-free medium containing the test compounds. Scratches were evaluated microscopically (Motic AE 2000 inverted microscope, Barcelona, Spain), and three images of each scratch were recorded using a Moticam 2500 at defined time points: 0, 20 and 30 h. Cell migration was measured in Motic Images PLUS v2.0 software by calculating scratch closure, given as the total migrated area after treatments in relation to the initial scratch area at 0 h (considered as 0% wound closure).
Ae2000 inverted microscope
Manufactured by Motic
Sourced in Hong Kong
The Motic AE2000 Inverted Microscope is a compact and robust microscope designed for laboratory use. It features a reversed optical path, with the objective lens positioned below the specimen stage, allowing for easy access and observation of samples. The microscope is equipped with a high-quality optical system, providing clear and detailed images. The AE2000 is suitable for a variety of applications, including cell culture monitoring, tissue examination, and other life science research tasks.
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14 protocols using ae2000 inverted microscope
1
Breast Cancer Cell Migration Assay
2
Oxidative Stress Assay in Neuro-2A Cells
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Neuro-2A cells were seeded in 6-well culture plates at a density of 80 × 104 cells/well and incubated at 37 °C for 24 h. Cells were incubated with various concentrations (50 to 200 µM) of H2O2 and photographed 24 h later using a Motic AE2000 Inverted Microscope (Motic, Hong Kong, China).
3
Matrigel Invasion Assay for Prostate Cancer
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WT and LMPTP KO MyC-CaP and C4-2B PCa cells were serum starved overnight (18 hours) in DMEM or RPMI media containing 0.1% FBS with 1X Pen/Strep. Cells were placed in the top chamber of a Matrigel Invasion Chamber (Corning, #354480) with the bottom chamber containing DMEM or RPMI media with 10% FBS with 1X Pen/Strep to promote migration. For experiments with LMPTP inhibitor, 10 μM Compd. 23 or DMSO was added to the top chamber. Two days later, cells on the bottom of the transwells were fixed with 100% methanol and stained using 0.05% crystal violet in 25% ethanol. Migrated cells were imaged using the Motic AE2000 Inverted Microscope and counted from 5 non-overlapping frames.
4
Wound Healing Assay with Ibrutinib
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Cells were seeded in 6-well plates and cultured until reaching 90% confluence. After, cells were wounded using a sterile pipette tip and serum deprivation conditions were imposed. Then, cells were treated with the IC50 dose of Ibrutinib and the wounded area was photographed at 0, 3, and 5 days using a Motic AE2000 Inverted Microscope (×100 magnification). The area of the scratch was measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA, version 1.53o), and the analysis was performed by comparing the initial area opened by the scratch at the initial time point with the area still open at each timepoint.
5
3D Colony Formation Assay for Prostate Cancer
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A 1:1 ratio of 1.2% w/w noble agar (VWR, #90000–772) to 2X DMEM or RPMI media was plated on the bottom of 6 well plates. After solidification of the agar, MyC-CaP or C4-2B PCa cells were suspended in a 1:1 mixture of 0.6% noble agar to 2X DMEM or RPMI media and plated on top (35 K MyC-CaP cells/well and 20 K C4-2B cells/well). After solidification of the top layer, growth media with 10% FBS was added to each well to prevent gel agar from drying. Plates were maintained at 37°C and 5% CO2 while maintaining a thin layer of growth media. After 21 days, colonies were stained with crystal violet and washed. Colonies were visualized and counted throughout the depth of the agar using the Motic AE2000 Inverted Microscope. Representative images were captured using the AxioVert Marianas Microscopy System as Z-stacks and compiled into a single image using ImageJ (55 (link)).
6
UNC0646 Inhibits Cell Migration
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The effect of UNC0646 on cell migration was determined by wound scratch assay, according to Liang et al. [26 (link)]. In brief, cells were seeded in 6-well plates and cultured until reaching 90% confluence. Then, serum deprivation conditions were imposed and the cell monolayers were gently scratched across the center of the well using a 200 μL pipette tip. The detached cells were removed by PBS washing. At this time, cells were treated with the IC25 dose of UNC0646 for 24h or 48h. Scratch closure was monitored and imaged at 0, 24 h, and 48 h using a Motic AE2000 Inverted Microscope (×100 magnification). The area of the scratch was measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the analysis was performed by comparing the initial area opened by the scratch at the initial time point (0 h) with the area still open at each timepoint.
7
In Vitro Wound Healing Assay
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The in vitro wound-healing assay was optimized according to Liang et al. [14] (link). MCF7 cells and MDA-MB-231 cells were seeded in 24-well plate with an inoculum of 2 × 105 cells per well and cultured in complete media for 24 h. After 24 h, media was removed, and each well was scratched using a 200 μL pipette tip, leaving a gap of approximately 0.8 mm in width. Cells were washed twice with PBS to remove detached cells and cell debris. Cells were kept in FBS- and insulin-free media containing the test compounds. The distance between the two limits of the scratch was monitored using a Motic AE 2000 inverted microscope with an objective of 10 × at 0 and 24 h after compounds addition. Images were collected using a Moticam 2500 and measurements were performed using Motic Images Plus V2.0 software. Zero h was considered as 0% of wound closure. At each time point one photo of each scratch was taken and three representative measures were performed. Each assay was performed with intern triplicates and at least 4 independent experiments were performed per condition.
8
Giemsa Staining of Cells
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After removing culture medium, cells were fixed with methanol and washed with deionised water before immersing them in Giemsa stain for 15 min at RT. After another wash with deionised water, cells were examined under a MOTIC® AE 2000 inverted microscope, Kowloon, Hong Kong.
9
Cell Migration and Invasion Assay
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For the migration assay, MIA PaCa-2 and Panc-1 cells (5.0 × 104 cells per well) were plated in the top chamber inserts with polyester membrane (8 μm pore size) of 24-well plates (Corning, New York, NY, USA) overnight. The bottom chambers of the plates were filled with DMEM containing 10% (v/v) FBS. The various drug treatments, suspended in serum-free DMEM, were added to the upper chamber, and 0.75 mL 10% (v/v) FBS medium was added into the lower compartment. After 24 h of treatment, cells in the top chamber were removed using a cotton swab, and cells that migrated to the bottom chambers were fixed in methanol and stained with 0.1% (w/v) crystal violet for 0.5 h at room temperature. The stained cells were counted in 3 independent fields under an AE2000 inverted microscope (Motic, Carlsbad, CA, USA). For the invasion assay, a similar protocol was followed apart from the replacement of the top chamber of the transwell plate coated with 50 μL Matrigel (Corning, New York, NY, USA).
10
Radial Migration of Spheroids in 3D
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Radial migration was performed in 3D cultures according to Vinci et al. [26 (link)]. Flat-bottomed, 24-well plates (Sarstedt, NC, USA) were coated with 0.01 mg/mL Poly-D-Lysine (Sigma-Aldrich®, St. Louis, MO, USA) in sterile water for 2 h at room temperature, followed by 1:30 diluted MatrigelTM coating for 30 min. Four-day spheroids were transferred to pre-coated plates and maintained in 500 μL of medium containing 10% FBS, 2% MatrigelTM and the compounds, [15]pyN5, [16]pyN5 and ARP-100, in concentrations ranging from 5–40 µM. Images were captured at 5, 10 and 24 h using AE 2000 inverted microscope (Motic, Hong Kong, China) and the area between cells in the migration front and the perimeter of the spheroid measured. More than 10 spheroids per condition were analyzed.
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