MDA-MB-231 cells were seeded in a six-well plate, and growing until forming confluent monolayer. A ∼300 μm-wide linear wound was made using a sterile 10 μl pipette tip. The suspend cells were washed away with PBS buffer. The remaining cells were cultured in DMEM medium containing 0.1% FBS. Cells were allowed to close the wound for 48 h and the wound areas were photographed with the Live Cell Imaging System (Leica DMI600B) every 15 min.
Live cell imaging system
Manufactured by Leica
Sourced in Germany
The Live Cell Imaging System is a microscopy-based instrument designed for real-time observation and analysis of living cells. It provides a controlled environment for cells to be imaged over extended periods, enabling researchers to study dynamic cellular processes.
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5 protocols using live cell imaging system
1
Wound Healing Assay with MDA-MB-231 Cells
2
Evaluating Cell Invasiveness and Migration
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H1299 cell invasiveness was assessed utilizing 24-well transwell chambers (8μm, Corning, Lowell, MA, USA). In a succinct sequence, following a 24-hour incubation period, the chambers underwent cleansing with cotton swabs, fixation with 4% paraformaldehyde for 20 minutes, and subsequent staining with crystal violet. The enumeration of cells was conducted in three randomly selected fields within each chamber, and the resultant values were averaged.
For the evaluation of migratory potential, a single-cell suspension was introduced into a 6-well plate and cultivated until cells reached 90%-100% confluency. Subsequently, a controlled and vertical scratch was generated using a 200 µl pipette tip, creating a wound. Detached cells were systematically purged with PBS, and the medium was subsequently replaced with 1 ml of serum-free medium. The 24-well plate was positioned in the Live Cell Imaging System (Leica, Brunswick, Saxony, Germany), capturing images of the wound at both 0 h and 24 h. Measurements of wound distances were taken, and the rate of wound healing was evaluated.
For the evaluation of migratory potential, a single-cell suspension was introduced into a 6-well plate and cultivated until cells reached 90%-100% confluency. Subsequently, a controlled and vertical scratch was generated using a 200 µl pipette tip, creating a wound. Detached cells were systematically purged with PBS, and the medium was subsequently replaced with 1 ml of serum-free medium. The 24-well plate was positioned in the Live Cell Imaging System (Leica, Brunswick, Saxony, Germany), capturing images of the wound at both 0 h and 24 h. Measurements of wound distances were taken, and the rate of wound healing was evaluated.
3
Fluo-4 Ca2+ Imaging in HepG2 Cells
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HepG2 cells were cultured in a 6-well plate for 24 h and then incubated by hank’s balanced salt solution (HBSS, without Ca2+ and Mg2+) containing 1 μmol/L fluo-4/AM and 0.5‰ pluronic F-127 for 20 min at incubator. After fluo-4/AM loaded, the cells were washed by HBSS for three times and then equilibrated in HBSS for 30 min in the dark. The fluorescence intensity of fluo-4-Ca2+ complex was detected by a Live Cell Imaging System (AF7000, Leica, Germany) with Ex 488 nm and Em 516 nm, and 20 mmol/L KCl was added into wells at almost 15 s. The fluorescence curve was calculated by ∆F/F0 = (the mean fluorescence intensity of cells – the background intensity)/the background intensity.
4
Wound Healing and Calcium Signaling Assays
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The wound healing assays were applied for assessing the migration capacity of cells. Cells were seeded into 6-well plates with complete medium, at a density of 5 10 5 cells/well and then cultured for 24 h. The linear wounds were scratched by a pipette tip on the completely con uent monolayer cells, timed 0 h and then cells were cultured in serum free medium for 24 h, the last moment timed 24 h. The images at 0 h and 24 h were captured by an inverted microscope and the migration % = (the distance of two sides at 0 h (D0) -the distance of 24 h (D24))/D0. 2.8 Fluo-4-Ca 2+ complex analysis HepG2 cells were cultured in a 6-well plate for 24 h and then incubated by hank's balanced salt solution (HBSS, without Ca 2+ and Mg 2+ ) containing 1 μmol/L uo-4/AM and 0.5‰ pluronic F-127 for 20 min at incubator. After uo-4/AM loaded, the cells were washed by HBSS for three times and then equilibrated in HBSS for 30 min in the dark. The uorescence intensity of uo-4-Ca 2+ complex was detected by a Live Cell Imaging System (AF7000, Leica, Germany) with Ex 488 nm and Em 516 nm, and 20 mmol/L KCl was added into wells at almost 15 s. The uorescence curve was calculated by ∆F/F 0 = (the mean uorescence intensity of cells -the background intensity)/the background intensity.
5
Calcium Imaging in HepG2 Cells
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HepG2 cells were cultured in a 6-well plate for 24 h and then incubated by hank's balanced salt solution (HBSS, without Ca 2+ and Mg 2+ ) containing 1 µmol/L uo-4/AM and 0.5‰ pluronic F-127 for 20 min at incubator. After uo-4/AM loaded, the cells were washed by HBSS for three times and then equilibrated in HBSS for 30 min in the dark. The uorescence intensity of uo-4-Ca 2+ complex were detected by a Live Cell Imaging System (AF7000, Leica, Germany) with Ex 488 nm and Em 516 nm, and 20 mmol/L KCl was added into wells at almost 15 s. The uorescence curve was calculated by ∆F/F 0 = (the mean uorescence intensity of cells -the background intensity)/the background intensity.
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