In addition to the wet lab practical set up, ensure you have microscopes that are either connected to the network or that you have a USB memory stick for each microscope. Create the shared folders on your network to allow student groups to access the images as soon as possible. You can either take images of stage micrometres at each magnification and add to the shared folder or if there is time ask each group to take an image of the slide micrometre at the appropriate magnification. Ensuring adequate support in the first imaging session ensures a much smoother process and better results in future sessions.

Common errors to look out for include:

• • Inconsistent scratches:
    • encourage students to use their spare hand to hold the wrist of the hand making the scratch to reduce shaking,
    • students should be told to consider the angle at which they are holding the pipette tip to keep the width consistent between wells.
  • Inaccurate pipetting:
    • try to encourage students to perform serial dilutions where needed to avoid pipetting small amounts e.g. 1 µl.
  • Imaging errors:
    • ensure students note the magnification of the microscope
    • encourage students to adjust the focus for a clear image
    • ensure students take images where either the centre of the cross or the marker are visible to enable them to image the same place again at 4 hrs
    • ensure students marks are on the underside of the wells not on the lids otherwise the marks are not in focus for the images
    • ensure students don’t make marks directly under the scratch in the same plane otherwise students cannot see the scratch properly
    • ensure students label their images properly to avoid mix ups

• • Measurement errors:

• • • encourage students to think logically about the size of the scratch and whether their measurement seems feasible.
    • if measurements do not seem feasible, check FIJI is calibrated correctly to convert pixels into micrometres
    • discuss with each group how they are taking the measurements, sometimes different people within the same group are measuring using different reference points.

The following are common discussion points that are worth having with students when they are planning their experiments:

• • How many repeats of each condition are you having?
    • We encourage at least 3 repeats of any condition and discuss that this is too few but OK given their limited well numbers. It’s possible here to open up a discussion about biological repeats and the fact that these cells were all plated from the same batch.
  • What controls are you using?
    • Many students think that because they completed controls in labs 1 and 2, they do not need to complete more controls. We teach them to always have controls.
    • The concept of DMSO as a negative control confuses some students. Explain about the drugs being dissolved in DMSO and how they need to check that any effects seen are due to the drug and not the DMSO.
  • What concentration are you using?
    • Some students just use the recommended concentration without thinking why or want to use quite random concentrations. We use this as an opportunity to discuss IC50 and why you don’t want to use a high concentration that starts to inhibit other channels as well. You can also use it to discuss choosing concentrations that work well on a semi-log graph.

References

Gao, R., Shen, Y.,  Cai, J., Lei, M., Wang, Z. (2010) Expression of voltage-gated sodium channel alpha subunit in human ovarian cancer. Oncology reports, 23, 1293-1299. https://doi.org/10.3892/or_00000763

Liang, C., Park, A.Y., Guan, J. (2007). In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature Protocols, 2, 329–333.  https://doi.org/10.1038/nprot.2007.30

Mao, W., Zhang, J., Körner, H., Jiang, Y., Ying, S. (2019). The emerging role of voltage-gated sodium channels in tumor biology. Frontiers in oncology, 9(124). https://doi.org/10.3389/fonc.2019.00124