Cell counting kit (CCK-8; Dojindo, Kumamoto, Japan) assays were used to detect cell proliferation. Migration was examined by both a wound-healing assay and a Transwell assay (uncoated insert). A Transwell assay (Matrigel-coated insert) was used for invasion monitoring. Cell migration and invasion assays were performed using a Transwell technique with uncoated polycarbonate inserts (Millipore, Darmstadt, Germany) or BioCoat™ inserts (BD Biosciences, NJ, USA). Medium without FBS (2 × 104 cells/200 μL) was added to the upper portion of a migration (uncoated insert) or invasion (Matrigel-coated insert) chamber, with 600 μL of DMEM containing 10% FBS added to the lower chamber. All experiments were performed three times, and the cell numbers were counted at least three times to calculate an average.
Counting kit cck 8
Manufactured by Dojindo Laboratories
Sourced in Japan
The Counting kit (CCK-8) is a colorimetric assay used for the determination of cell viability and cytotoxicity. It employs a water-soluble tetrazolium salt which is reduced by living cells, producing a colored formazan dye that can be measured spectrophotometrically.
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2 protocols using counting kit cck 8
1
Cell Proliferation, Migration, and Invasion Assays
2
CCK-8 Cell Viability and Transwell Migration Assays
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Counting Kit (CCK)-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). 5x10 3 OS cells were seeded onto 96-well plates and incubated for 1, 2 and 3 days. Subsequently, 10 µl of CCK8 reagents were added to the 96-well plates, after 2 h incubation at 37˚C, the absorbance at 450 nm was measured to evaluate the number of viable cells by SUNRISE microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland).
In vitro migration and invasion assays. The transwell assay was conducted in 24-well BD Matrigel invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Briefly, 5x10 4 OS cells were seeded in the upper well of the migration chamber in DMEM without serum, and 500 µl DMEM supplemented with 10% FBS were added to the lower chamber well. After 24 h incubation, the cells on the top of the well were removed with a cotton swab, and the bottom cells were fixed with 4% paraformaldehyde, subsequently stained with 0.1% crystal violet for 30 min. Images were captured in 5 independent fields. For invasion assay, the membranes were coated with Matrigel (BD Biosciences).
In vitro migration and invasion assays. The transwell assay was conducted in 24-well BD Matrigel invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Briefly, 5x10 4 OS cells were seeded in the upper well of the migration chamber in DMEM without serum, and 500 µl DMEM supplemented with 10% FBS were added to the lower chamber well. After 24 h incubation, the cells on the top of the well were removed with a cotton swab, and the bottom cells were fixed with 4% paraformaldehyde, subsequently stained with 0.1% crystal violet for 30 min. Images were captured in 5 independent fields. For invasion assay, the membranes were coated with Matrigel (BD Biosciences).
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