Before the Experiment:
1.Prior to the experiment, check for flammable or explosive materials in the room and ensure the safety and reliability of the laboratory environment.
2.Wear safety protective gear, such as safety glasses, rubber gloves, and protective masks.
3.Inspect the laboratory bench for any debris, and clean all equipment and instruments meticulously with tweezers.
4.If necessary, arrange the experiment on the bench at an appropriate distance to avoid wasting valuable reagents.
5.Calibrate instruments before conducting the experiment.

Figure 1. safety training

During the Experiment: 1.Smoking is strictly prohibited in the laboratory to prevent fire hazards, and eating in the laboratory is discouraged to avoid contaminating equipment.
2.Wear laboratory attire, and remind students not to place mobile phones or personal items on the bench to prevent damage to equipment.

Figure 2. safety training2

1.Avoid repeated freezing and thawing of MasterMix; if used frequently, it is best to dissolve it and store it at 4 degrees Celsius.
2.Prepare more Mixes to reduce sampling errors, and perform operations on ice whenever possible.
3.Change the pipette tip for each tube or well; do not use the same tip continuously.
4.After adding all components, centrifuge to remove air bubbles.
5.Include at least three replicates for each sample.

1.Avoid the formation of bubbles between the lower culture medium and the insert. Bubbles can weaken or eliminate the chemotactic effect of the lower culture medium. When placing the insert into the culture plate, pay attention, and if bubbles form, lift the insert, remove the bubbles, and then place it in the culture plate.
2.The choice of time points for the experiment depends on the invasion ability of cancer cells, treatment factors, and other considerations.
3.When wiping the upper chamber cells with a cotton swab, be careful not to touch the layer of cells that has penetrated the membrane.
4.For cells that are difficult to pass through the membrane, consider serum starvation for 12-24 hours before starting the experiment.

Sterile Operations:
1.Perform experiments in a sterile workstation or sterile environment to ensure aseptic conditions.
2.Before conducting experiments, disinfect the bench surface with a disinfectant and place the required culture equipment inside.
3.Before conducting experiments, disinfect hands with 70% ethanol and wear sterile gloves.
4.When opening and sealing culture dishes, minimize exposure to air to prevent bacterial or microbial contamination.

Cell Culture Conditions:
1.Select appropriate culture media based on cell type and prepare the media according to the manufacturer's instructions.
2.Control the culture temperature and CO2 concentration to promote cell growth and division based on cell type and medium requirements.
3.Regularly check the pH and osmotic pressure of the culture medium and adjust as needed.
4.Avoid over-confluence or sparse cell density. Choose an appropriate cell density based on cell growth rate and dish size.

Cell Passaging:
1.Before cell passaging, check the health and growth status of the cells.
2.If cells show abnormalities or contamination, replace the culture medium or re-culture the cells.
3.Use appropriate cell detachment reagents or enzymes to separate cells and follow the manufacturer's instructions and recommendations.
4.When passaging cells, avoid over-dispersion or aggregation to maintain cell stability and functionality.

Figure 3.Ultra-Clean Workbench

Figure 4. Ultra-Low Temperature Freezer