For invasion assays, 1.0×105 cells were seeded in a Matrigel-coated chamber (BD Biosciences). Cells were seeded in serum-free media and then cultured in complete growth media. After 24 hours of incubation at 37°C, cells that had invaded were fixed and stained in dye solution containing 20% methanol and 0.1% crystal violet. Invasive cells were imaged using a BH-2 inverted microscope (Olympus). The mean values of three duplicate assays for each experimental condition were used for statistical analysis.
Bh 2 inverted microscope
Manufactured by Olympus
Sourced in Japan, United States
The BH-2 inverted microscope is a laboratory equipment product manufactured by Olympus. The core function of this microscope is to provide a high-quality optical system for the observation and analysis of samples from the underside, allowing for a unique perspective on the specimen under investigation.
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4 protocols using bh 2 inverted microscope
1
Cell Invasion Assay Protocol
2
Matrigel Invasion Assay for Cell Migration
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For invasion assays, 5.0×104 cells were seeded in the upper well of a Matrigel coated chamber with serum-free media (BD Biosciences, Franklin Lakes, NJ, USA). Complete growth medium was added to the lower well of each chamber. After 48 h of incubation at 37°C, cells that had migrated or invaded to the lower well of the chamber were fixed and stained in dye solution containing 20% methanol violet and 0.1% crystal, and imaged using a BH-2 inverted microscope (Olympus, Tokyo, Japan). Cell counts are expressed as the mean number of cells per field of view and were normalized to the negative control group. Three independent experiments were performed and the data are presented as mean ± standard deviation (SD).
3
Apoptosis Induction Assay with AO
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Cells (5x104) cultured on cover-slides in 24-well plates were incubated for 14 h in RPMI 1640 medium containing 2% fetal calf serum, preincubated with sulindac sulfide for 6h, and then treated with or without β-lapachone for 24 h, then were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature (RT), and stained for 10 min with 0.5 ml of AO (10 mg/ml in PBS) (Sigma). After several PBS washes, the cells were examined on an Olympus BH-2 inverted microscope equipped with a fluorescence attachment.
4
Oil Red O Staining of Lipid Droplets
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MDA-MB-231 and BT549 cells were plated in 6-well plates at 5 × 104 cells/mL, and allowed to adhere overnight before replacing media with fresh media ±1 µg/mL LT-IIc. Cells were treated for 24 h followed by fixation with 4% phosphate buffered formalin, 2 mL per well, for 10 min. Wells were rinsed with distilled water, then 60% isopropanol for 2–5 min. Fresh Oil Red O (Sigma, Cat.# O-0625) was prepared immediately from a stock solution of 0.3% Oil Red O (w/v) in 99% isopropanol. Immediately before use, the 0.3% Oil Red O solution was further diluted at a 3:2 ratio with distilled water and filtered using #1 Whatman filter paper. Oil Red O was applied for 5 min, followed by 3× rinses with 2 mL/well of dd H2O. Cells were photographed in situ in the plate using an Olympus BH2 inverted microscope (Waltham, MA, USA) with a Moticam 2.0 digital camera, under bright field illumination to see Oil Red O, and phase contrast, to visualize cellular vacuoles.
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