I am using T47Ds, HCC1937s, and SKBR3s. I am plating around 3,000 cells/well in 100 uL in a 96 well plate. I had been told it was not enough to wait 24 hours after plating to treat the cells and that 36 would be better. Anyone have any advice?
Hi Marie,
I doubt it makes much difference. If the cells have stuck down and spread out after 24h and look as if they're growing normally, I'd say you can go ahead with the treatment. I do quite a lot of similar experiments in 96-well plates with dosing 24h after seeding, without any problems. To put your mind at rest, you could do it either way at first to see if the IC50s come out any different, but I'd be surprised if there was any significant difference.
Usually we assume that after plating. cancer cells will go through a latent period before they can duplicate again. In this period, whose lenght can vary according to different cell types and experimental conditions, they are usually insensitive to most anticancer agents that kill only or prevalently the proliferating cells. So to calculate properly the IC50 of a drug, we have to treat the cells after they enter the exponential phase of cell growth. My advice is to build in advance the growth curve of each cell line in your experimental system in the absence of any drug and then choose the proper time for adding the drug (exponential phase) that can be 24, 48, 72 or more hours after plating. Furthermore, 3,000 cells/well could be too little, for the MTS assay for example 10,000-50,000 cells/well could be a more appropriate number to seed. Of course the number of cells to plate depends on the sensitivity of the method employed to measured cell survival. Hope to be useful. Regards,
24 h and 36 h timeline have been based on confluency and health of cells. In most of the cases, 24h gives sufficient time to adhere and form a monolayer. Depending upon the number of passages, cells may behave differently and take a longer time to achieve 95% confluency ( usually recommended for IC50). Thus, it would be wise to go with confluency and health of cells rather than a timeline of 24 or 36h.
24h is sufficient to stabilize the cells. You may need to increase the number of cells to 5000/well for better communication. 50 to 60% confluent is good for treatment.
In my opinion, If you start with 10,000 cells to 30,000 cells/100uL in 96 well plate, 24 h is sufficient time if cells are healthy. Before seeding, check the cell viability using trypan blue or any other methods. In case of sensitive cells like HCC1937 , seed around 10,000- 25000 cells/ 100uL per well. If your cells are not attaching or getting washed away while washing with PBS due to high passage number use longer incubation time >24 . It is advisable to use cells from 3-6 passage after recovery. After-that, cells start loosing its adherence property and morphology.Subculture cells when it is about 85% to 95% confluent. For IC 50 experiment, treat the cells with drugs once they are properly attached and incubate the cells for 24 h ,48 h and 72 h.
When cells are about 50-60% confluent is good time for treatment for 24h. If you need longer treatment you may stat at 20% then continue up to 72h.
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