Briefly, cells (1 × 104 cells) were exposed to different concentrations of HL156A in 1 mL of 0.3% basal medium Eagle's agar containing 10% FBS and were plated in 60‐mm plates containing 0.6% low‐melting temperature agarose. The cells were allowed to form colonies for 14 days at 37°C in a humidified atmosphere containing 5% CO2. The colonies were fixed with 4% paraformaldehyde for 30 min at room temperature and were then stained with 0.04% crystal violet for 30 min at room temperature. The cell colonies were scored using an IX2‐SLP inverted microscope (Olympus, Tokyo, Japan).
Ix2 slp inverted microscope
Manufactured by Olympus
Sourced in Japan
The IX2-SLP is an inverted microscope designed for live-cell imaging and observation. It features a stable, vibration-resistant frame and supports a variety of objectives and stages. The IX2-SLP is intended for use in research and laboratory settings.
Automatically generated - may contain errors
7 protocols using ix2 slp inverted microscope
1
Colony Formation Assay with HL156A
2
Heat Shock Modulates hDPC Migration
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The cells were allowed to grow in a culture dish overnight, and a scratch approximately 3 mm in width was created in the monolayer using a pipette tip. After washing twice with PBS, the cells were subjected or not subjected to heat shock (42°C for 30 min), and images were captured after 24 h. The migratory behavior of the hDPCs was assessed using a transwell migration assay (Chemotaxis Cell Migration Assay kit, Chemicon). hDPCs were seeded onto Thincert™ tissue culture inserts (pore size = 8 μm; Greiner Bio-One, Frickenhausen, Germany) in DMEM with 10% FBS at a density of 1.5 x 105 cells per insert. The cells were subjected or not subjected to heat shock and allowed to migrate for 24 h. Under some conditions, inhibitors against Pin1 and MAPKs were also added to the hDPCs. After 24 h, the hDPCs that had transmigrated towards the lower surface of the filter were fixed with methanol, stained with hematoxylin for 5 min and washed with PBS. The cells in five random microscopic fields per well were imaged using an Olympus IX2-SLP inverted microscope (Japan) at 100X magnification. The numbers of migrated cells on the lower side of the membrane were counted in five randomly selected areas, and the values are expressed as the mean area percentage.
3
Cell Migration Assay with HL156A
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The cells were plated at a density of 1 × 106 cells in a 6-well plate and allowed to grow in a 5% CO2 incubator at 37 °C for 24 h before a scratch ~3-mm wide was created in the cell monolayer using a pipette tip. After being washed twice with PBS, the cells were incubated without or with HL156A. The cells were imaged in 4 random microscopic fields per well using an Olympus IX2-SLP inverted microscope (Olympus, Japan) at ×100 magnification. The distance migrated by the cell monolayer was measured during indicated period, in order to close the wounded area. The distance was determined using ImageJ software.
4
Cell Migration Assay with HL156A
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Cell migration was investigated using a two-chamber migration assay with a pore size of 8 mm. In total, 2 × 106 cells were treated with or without HL156A before being resuspended in 200 µL of serum-free medium and seeded on the upper compartment of a 12-well Transwell culture chamber. An amount of 700 µL of complete medium (Welgene, Daegu, Korea) was added to the lower compartment. After incubation at 37 °C, the chambers were washed twice with PBS to remove nonattached cells, fixed in 4% paraformaldehyde solution for 2 min at room temperature and stained with 0.05% crystal violet diluted in 20% methanol for 10 min at room temperature. Images of migrated cells were captured using an Olympus IX2-SLP inverted microscope (Olympus, Japan).
5
Soft Agar Colony Formation Assay
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In brief, the cells (1 × 104 cells/mL) were suspended in 1 mL of 0.3% basal medium Eagle’s agar containing 10% FBS and were plated in 60-mm plates containing 0.5% low melting temperature agarose. A layer of medium was added to the agar surface to prevent aridity. The cells were allowed to form colonies for 14 days at 37 °C in a humidified atmosphere containing 5% CO2. The colonies were fixed with 4% paraformaldehyde for 15 min at room temperature and were then stained with 0.005% crystal violet (Sigma, St. Louis, MO, USA) diluted in 20% methanol for 30 min at room temperature. The cell colonies were scored using an IX2-SLP inverted microscope (Olympus, Tokyo, Japan).
6
Scratch Wound Healing Assay
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Cells were allowed to grow in a culture dish overnight and a scratch ~3 mm wide was created in the monolayer using a pipette tip. After being washed twice with PBS, the cells were treated with or without HL156A, and images were captured after 24 hours. Cells were imaged in 5 random microscopic fields per well using an Olympus IX2‐SLP inverted microscope (Olympus) at ×100 magnification.
7
Cell Cycle Analysis and Soft Agar Assay
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The cell cycle distribution was analyzed by flow cytometry. Briefly, 1x10 6 cells were harvested and washed in PBS, and then fixed in 70% alcohol for 30 min at 4˚C. After washing three times in cold PBS, the cells were resuspended in 1 ml of PBS solution containing 50 µl of 1 mg/ml PI and 1 unit of DNase-free RNase for 30 min at 37˚C. The samples were then analyzed for their DnA content by FACS (Beckman Coulter Inc.).
Soft agar colony formation assay. Briefly, the cells (8x10 3 cells/well) were exposed to different concentrations of EX527 or AGK2 in 1 ml of 0.3% basal medium Eagle's agar containing 10% FBS. The cultures were maintained at 37˚C in a 5% CO 2 incubator for 10-15 days, and the cell colonies were scored using an Olympus IX2-SLP inverted microscope (Japan).
Soft agar colony formation assay. Briefly, the cells (8x10 3 cells/well) were exposed to different concentrations of EX527 or AGK2 in 1 ml of 0.3% basal medium Eagle's agar containing 10% FBS. The cultures were maintained at 37˚C in a 5% CO 2 incubator for 10-15 days, and the cell colonies were scored using an Olympus IX2-SLP inverted microscope (Japan).
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