For wound-healing assays, 0.5 × 106 cells were seeded in 60-mm dishes and cultured until confluent. Using a (yellow) pipette tip, a straight scratch (wound) was generated, keeping the pipette tip at an angle of ~30°. Wound closure was then monitored over 48 h. For invasion assays, QCM 24-Well Cell Invasion assay kit was used (Millipore, Burlington, MA, USA), according to manufacturer’s guidelines. Briefly, insert interiors were rehydrated with pre-warmed serum-free media, and 0.3 × 106 cells were added to each insert. Complete media (10% FBS) was added to the lower chambers, and cells were incubated for 48 h in a CO2 incubator. Cells were stained with 0.1% crystal violet solution, and images were obtained using the 4× or 10× objective.
Qcm 24 well cell invasion assay kit
Manufactured by Merck Group
Sourced in United States
The QCM 24-well cell invasion assay kit is a laboratory equipment product designed to measure the invasive capabilities of cells. It provides a standardized platform for evaluating the ability of cells to migrate through a basement membrane-like barrier. The kit includes a 24-well cell culture plate, inserts with a microporous membrane, and a dye-based detection system. This product enables researchers to quantify and compare the invasive properties of different cell types or assess the effects of various treatments on cell invasion.
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7 protocols using qcm 24 well cell invasion assay kit
1
Wound Healing and Invasion Assays
2
Cell Invasion Assay Using QCM Kit
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The cell invasion assay was performed using the QCM 24-well cell invasion assay kit (Millipore, USA). Each lower chamber contained an additional 600 μl of 0.5% FBS as the chemoattractant. Cells (1 × 105) were placed into the upper chamber and then incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2. After incubation, non-migrating cells in the top chambers were completely removed with a cotton bud. Cells that invaded into the lower chambers were fixed in 95% methanol for 15 min, stained with 0.1% crystal violet for 10 min prior to washing with water, and counted in five random fields with an inverted microscope.
After dyeing, the lower membrane was decolorized with 33% acetic acid, and the crystal violet was completely eluted. The eluent was measured with a spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Each assay was replicated three times.
After dyeing, the lower membrane was decolorized with 33% acetic acid, and the crystal violet was completely eluted. The eluent was measured with a spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Each assay was replicated three times.
3
Quantification of Cell Invasion
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Cell invasion was measured using the QCM™ 24-well Cell Invasion Assay Kit (Millipore), according to the manufacturer’s instructions. Briefly, cells were seeded onto insert chambers containing a collagen-coated polycarbonate membrane with 8-μm pores. Cells that invaded the ECM layer were stained with 4′,6-diamidino-2-phenylindole (DAPI). Invading cells in five fields per chamber were visualized and counted under the LSM700 Confocal Laser Scanning Microscope (Carl Zeiss, Jena, Germany). Each experiment was performed three times independently.
4
Cell Invasion Assay Using QCM Kit
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The cell invasion assay was performed using the QCM 24-well cell invasion assay kit (Millipore, USA). Each lower chamber contained an additional 600 μl of 0.5% FBS as the chemoattractant. Cells (1 × 105) were placed into the upper chamber and then incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2. After incubation, non-migrating cells in the top chambers were completely removed with a cotton bud. Cells that invaded into the lower chambers were fixed in 95% methanol for 15 min, stained with 0.1% crystal violet for 10 min prior to washing with water, and counted in five random fields with an inverted microscope.
After dyeing, the lower membrane was decolorized with 33% acetic acid, and the crystal violet was completely eluted. The eluent was measured with a spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Each assay was replicated three times.
After dyeing, the lower membrane was decolorized with 33% acetic acid, and the crystal violet was completely eluted. The eluent was measured with a spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Each assay was replicated three times.
5
Cell Invasion Assay Using QCM Kit
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Invasion assay was performed using QCM 24-well cell invasion assay kit (Millipore, Boston, MA) according to the manufacturer's instructions. Briefly, cells were starved for 24 h prior to analysis. Cells were harvested using the 5 mL Harvesting Buffer per 100 mm dish and were then collected and re-suspended in 1-5 mL Quenching Medium. 1.25×105 cells were seeded in the insert containing serum free media while the serum containing media were added to the lower chamber. Cells were incubated for 24 h at 37°C in a CO2 incubator. Cells that invaded through the ECMatrix-coated membrane were lysed in Lysis Buffer/Dye Solution and incubated for 15 min at room temperature. RFU values were read with a fluorescence plate reader using 480/520 nm filter set.
6
Cell Invasion Assay with 5-FU and Compounds
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The QCM 24-well cell
invasion assay kit (Millipore, Burlington, MA, USA) was used according
to the manufacturer’s guidelines. Briefly, the inset interiors
were rehydrated with prewarmed serum-free media, and 0.2 × 106 cells were added to each insert. The complete medium (10%
FBS) with 5 μM 5-FU, compound4 , or compound 5 was added to the lower chambers, and the cells were incubated
for 24 h in a CO2 incubator. The cells were then stained
with a 0.5% crystal violet solution, and images were obtained using
the 10× objective of an Olympus BX51 microscope (Olympus, Tokyo,
Japan) and an IX71 camera.
invasion assay kit (Millipore, Burlington, MA, USA) was used according
to the manufacturer’s guidelines. Briefly, the inset interiors
were rehydrated with prewarmed serum-free media, and 0.2 × 106 cells were added to each insert. The complete medium (10%
FBS) with 5 μM 5-FU, compound
for 24 h in a CO2 incubator. The cells were then stained
with a 0.5% crystal violet solution, and images were obtained using
the 10× objective of an Olympus BX51 microscope (Olympus, Tokyo,
Japan) and an IX71 camera.
7
Cell Invasion Assay Using QCM
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Invasion assay was performed using QCM 24-well cell invasion assay kit (Millipore, Boston, MA) according to the manufacturer’s instructions. Briefly, cells were starved for 24 h prior to analysis. Cells were harvested and re-suspended in 1 ml Quenching Medium. 1.25 × 105 cells were seeded in the insert containing serum free media while the serum containing media were added to the lower chamber. Cells then were incubated for 24 h in a CO2 incubator. Cells that invaded through the ECMatrix-coated membrane were lysed in Lysis Buffer/Dye Solution and incubated for 15 min at room temperature. RFU values were read with PerkinElmer’s EnSpire Multimode plate reader (Waltham, MA) in fluorescence mode using 480/520 nm filter set.
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