Cell plating density - (Sep/13/2010 )

I am working with SNO human esophageal carcinoma cells, and my general protocol includes plating at a density of 3x10^5 cells/ml in 35x10mm dishes or 6-well plates. After plating I usually incubate for 24 hours, following which I treat the cells with whatever treatment and incubate for another 24 hours before starting assays. My question is as follows: should 80-90% confluency be reached within the first 24 hours, or will that adversely affect my results? Currently 80-90% confluency is reached, and I was wondering whether this would be a problem for assays such as Alamar blue (should I therefore decrease the plating density?)
Also, I seem to be having problems with uneven cell attachment after plating. The cells tend to attach in high density to the center of the plate, with fewer cells seen at the periphery of the plates. Is there any way I could get a more even distribution on a plate?

I'm not certain if you mean 24hrs after treatment has been added or 24hrs after the cells have been plated. Typically, you should see the same pattern even if you plate out few cells, which I would do to convince myself that the cell number is not interfering the assay results. It also depends on when you are reading the results of the experiment. However, if the cells are 80-90% confluent at the end of treatment, the only problem I foresee is in the case that a particular treatment stimulates growth it will be underestimated by the assay.

EVEN PLATING : This can be accomplished by either adding cells dropwise in a circular motion over the wells or simply swirling the plate gently to even disperse the cell throughout the well after you have added cells and medium to the plates.

MJD on Mon Sep 13 15:03:06 2010 said:

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