After infecting HT1080 cells with shScr, shCol18a1#3, and shCol18a1#4 viruses made from the pKLO.1 vector and selecting with puromycin, I will seed them in preparation for a proliferation and migration assay.
For the proliferation assay, I will seed 40,000 HT1080 cells in three 6cm plates for each sample, resulting in nine total plates. I will count one plate of each sample on days 4, 5, and 6 to get the growth curve.
For the migration assay, I will need a single-cell confluent layer for a scratch test, or wound healing assay. I will use a 12-well plate, leaving several wells for each sample. I will vary the densities between the wells, ranging from 250,000 to 500,000 cells. Thus, I can begin the assay as soon as the cells adhere to the plate.
Are these seeding densities reasonable for my experiment? I appreciate any advice.
Hello Eric Liu
The doubling time HT1080 is around 24-26 hrs. So, for the proliferation assay, you will have to seed cells in such a way that after 6 days (final time point) they should not turn overconfluent. This will have an impact on the end results as overconfluent cells in the 6cm plate may begin to die due to lack of growth space and nutrients.
Therefore, for the proliferation assay, you will have to perform a pilot experiment where you may seed different cell densities keeping in mind that the cells should be 90-95% confluent on the 6th day which is the last time point of your assay. Generally, for a multi-well plate, the proliferation assay is performed when the cells are 75-80% confluent so that after 72 or 96 hrs. time point, these cells are at 90-95% confluency and not overconfluent.
For the migration assay, you should begin the assay when the wells are 90-95% confluent. Check for the confluency using the microscope. Please note that 100% confluence is not recommended since many anchorage-dependent cells experience contact inhibition, which may alter their migratory capacity. Wells with less than 80% confluence will result in unusable photos because the image analysis software will interpret gaps between cells as part of the perimeter of the scratch. After the cells have reached 90% confluence, damage the cells as appropriate for the application.
You need to first run a pilot experiment with different cell densities as you mentioned for the 12-well plate, and note the time when the cells reach 90-95% confluency, when the cells are attached and proliferating. I would recommend that you use a lower cell density so that the cells get enough time to attach and proliferate to reach 90-95% confluency.
Best.
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