3 replies to this topic

[AllenChiu]

I'm going to generate a growth curve with 4 slightly different A549 cell lines each infected with different mutant version of gene X.
I heard it's going to be complecated for counting.
And I do have some kind of Obsessive compulsive disorder when it comes to counting things.
Besides,I'm also warried about the dilution step which will add inaccuracy.
So I would like to know if I can turn to FACS for counting.
A question is should I count the whole amount of cell of each well or just a certain partition of it?
I think that the former will be more accurate since I'm not sure about if the cell will be Homogeneous during dilution.
Any advise from you is appreciated.
Thank you in advence!
PS:if I do have to count the cell number,should I use 24 well plate or 6 well ones?Is the cell amount per 24 well is enough for handling?

[bob1]

It shouldn't be too complicated to count all you have to do is the four samples and controls at each time point.  You don't need to count the whole volume of the cells at each time point, just a fraction of it, or you really will be there all day counting cells.  Basically you will need to set up a well or two per time point (more wells is more accurate, but also more work), then harvest the cells in the same fashion at each time point, count the cells and graph.

If you don't want to count cells you can do it by DNA presence (sybr green or DAPI or propidium iodide on a fluoresence plate reader) or MTT assay (absorbance).

[AllenChiu]

bob1, on 16 August 2010 - 03:16 PM, said:

It shouldn't be too complicated to count all you have to do is the four samples and controls at each time point.  You don't need to count the whole volume of the cells at each time point, just a fraction of it, or you really will be there all day counting cells.  Basically you will need to set up a well or two per time point (more wells is more accurate, but also more work), then harvest the cells in the same fashion at each time point, count the cells and graph.

If you don't want to count cells you can do it by DNA presence (sybr green or DAPI or propidium iodide on a fluoresence plate reader) or MTT assay (absorbance).

thank you very much,do you think MTT will as accurate as cell counting?

[bob1]

Manual cell counting has about a 15% error per chamber of a haemocytometer in the hands of an experienced user.  Counting by a coulter counter or FACS is probably more accurate.  I don't know the error on an MTT, but it should be consistent and is relatively quick and easy to do.