The gene I am studying is downregulated during adipocyte differentiation in 3T3-L1 cells. However, this gene is useful for cell proliferation before induction. Thus, if I am going to study its role in differentiation, I have to bypass the proliferation effect from cell seeding to post-confluency because the RNAi cells grow slower than the control cells. I plan to plate as many RNAi cells as control cells when confluent(-2d), then wait two days more. If I do this, the control cells will still grow for two more days. Alternatively, I could directly plate RNAi cells as many as control cells at 2d post confluent, eg, number of d0 control cells is 10,000, then i directly seed 10,000 control and RNAi cells. thus, two cells are both directly reach the 2d post-confluency condition, in the facet of cell number at least. If this alternative method is ok to use, would I still need wait two more days to let them be 2d post-confluent, if they still need some time to exit the cell cycle?
Unless you already know the doubling rate of the L1 cells with knockdown of your GOI, it will be difficult to determine how many cells to seed at day "-2" such that you'll have equivalent cell numbers to the control cells at day "0". Therefore, if you want to properly control for cell density effects at day "0", you'll have to choose from the following approaches:
1) perform a pilot experiment where you count the # of cells in the knockdown condition at day 0 starting from a known constant amount at day "-2", and in parallel assess control cells seeded at various initial seeding densities at day "-2", to determine what would be the appropriate matched condition for the control cells. I think it will work better to titrate down the number of control cells rather than try to overseed the knockdown cells (although in principle you can try both ways). Once you have conditions that
2) take a gunshot approach and simply seed varying number of control cells (decreasing initial cell densities) to see if any changes in differentiation efficiency of knockdown cells are mirrored by changes in total cell number in the controls.
3) invest the time to make an inducible knockdown system wherein you can initiate knockdown at either day "-2" or day "0". This would allow you to determine whether the important role for your gene takes place prior to induction with differentiation cocktail.
Although I have used the protocol in which you simply seed cells at high initial seeding densities and skip the two day growth to confluence step in the case of human MSCs, I have not seen this used for L1as, and would not expect it to work as well as the growth to confluence standard protocol.
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