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.
Uncoated polycarbonate inserts
Manufactured by Merck Group
Sourced in Germany
Uncoated polycarbonate inserts are a type of lab equipment used for various applications. They serve as a component or accessory in laboratory settings, providing a platform or container for samples, reagents, or other materials during experimentation or analysis. The core function of these inserts is to provide a sturdy and transparent enclosure for the containment and handling of laboratory specimens or solutions.
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7 protocols using uncoated polycarbonate inserts
1
Cell Proliferation, Migration, and Invasion Assays
2
Functional Assessments of lincRNA-p21
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HN6 and Cal27 cells were transfected with siRNA or plasmids of lincRNA-p21 for 24 h, and then seeded in the plates. As described in our previous study [25 (link)], cell proliferation experiments were performed using the Cell Counting Kit (CCK8; Dojindo, Kumamoto, Japan) assays. The colony-forming assay was performed to monitor the cloning capability of HN6 and Cal27 cells [22 (link)]. Cell migration and invasion assays were performed using a Transwell technique with uncoated polycarbonate inserts (Millipore, Darmstadt, Germany) or BioCoat™ inserts (BD Biosciences, Franklin Lake, NJ, USA). Medium without FBS (2 × 104 cells/200 μl) were added into the upper portion of a migration (uncoated insert) or invasion (matrigel-coated insert) chamber, with 500 μl DMEM containing 10% FBS added into the lower chamber. After crystal violet staining, the crossed cells were dissolved in 33% of acetic acid. OD 570 nm absorbance was measured.
3
Transwell Migration and Invasion Assay
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Cell migration and invasion assays were performed using Transwell technique with uncoated polycarbonate inserts (Millipore) for migration or BioCoat™ inserts (BD Biosciences) for invasion. Medium without FBS (1–5 × 104 cells/200 μL) was added into the upper chamber, with 500 μL DMEM containing 10% FBS added into the lower chamber. After crystal violet staining, the positive cells were counted and analyzed under microscope.
4
Analyzing Cell Migration and Invasion
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Transwell assays were performed to analyze cell migration and invasion. Uncoated polycarbonate inserts (Millipore) were used for the migration experiment, and BioCoat™ inserts (BD Biosciences, Franklin Lake, NJ, USA) were used for the invasion experiment. Transfected CAL27 and HN6 cells were seeded in the upper chamber. After 24 h, the cells were stained with crystal violet, and the number of crystal violet‐positive cells on the membrane was counted in three randomly selected fields of view at a magnification of 100×. For the wound healing assay, cells were seeded in 6‐well plates, and a 20‐μL pipette tip was utilized to scratch a uniform linear wound in the cell monolayer. Cell migration across the wound was observed at 0 h and 24 h after scraping, and fields of view were randomly selected and photographed.
5
Transwell Assay for Cell Migration
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Tumor cell migration and invasion were assessed through a Transwell assay. Uncoated polycarbonate inserts from Millipore were employed for migration, whereas for simulating an invasive environment, VitroGel RGD from TheWell Bioscience, North Brunswick, NJ, was used. In the upper chamber, 1-5 × 10 4 cells were cultured in an FBS-free medium. The lower compartment contained a culture medium. After incubation, cells that did not migrate on the upper membrane surface were removed while cells that moved to the lower chamber were fixed with 4% paraformaldehyde solution and stained with crystal violet solution, and subsequent quantification.
6
Cell Proliferation, Migration, and Invasion Assay
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Cell proliferation was detected using a Cell Counting Kit (CCK8; Dojindo, Kumamoto, Japan). Cells were seeded into 6-well plates at 500 cells/well for 10 ~ 14 days to analyze the clone-forming capacity. Transwell assays were performed using uncoated polycarbonate inserts (Millipore, Darmstadt, Germany) to test migration or BioCoat™ inserts (BD Biosciences, Franklin Lake, NJ, USA) to test invasion. EdU (RiboBio, Guangzhou, China) and TUNEL (Beyotime, Shanghai, China) staining were conducted according to the manufacturers’ protocols.
7
Assessing Tumor Cell Migration and Invasion
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We assessed the migration and invasion of tumor cells using a transwell assay. For migration, we used uncoated polycarbonate inserts (Millipore), while we used BioCoat™ inserts (BD Biosciences) for invasion. After starving the cells overnight, we filled the upper chamber with 1–5 × 104 cells suspended in DMEM without FBS and the lower chamber with 500 μL of DMEM containing 10% FBS. After crystal violet staining, we counted and analyzed the positive cells under a microscope.
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