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Extra info for Cell Growth, Differentiation and Senescence: A Practical Approach (Practical Approach Series)
Apply to a haemocytometer by pipetting from the edge of the coverslip and permitting diffusion by capillary action. Count cells from all samples within a short fixed time after mixing with trypan to obtain a constant and reproducible measure of viability. ) 3. The haemocytometer has counting chambers on two sides. Count both sides and average the numbers. 4. There are nine grids in a counting chamber. Count cells in as many grids as needed to count at least 100 cells/chamber. Count cells in symmetric grids to avoid over- or undercounting due to sedimentation of cells during diffusion.
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Continued 5. 1 mm (height)] and 2 is the trypan blue dilution. 2 Determining the doubling time The growth curve of cells in a tissue culture dish is S-shaped when plotted on linear co-ordinates and linear when plotted as the log of cell number vs. time on a linear scale. Take, for example, a hypothetical experiment in which 5 X 104 MDA-MB-435 human breast cancer cells were incubated in 100 mm tissue culture dishes in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum (FCS) supplemented with 2 mM glutamine with and without the presence of 1 ng/ml transforming growth factor B (TGF-B) and total viable cells were counted 1, 3, 5, and 7 days later.