I'm a cell biologist. I have a seemingly easy question for which I've received opposing answers.
I'm trying to compare the number of peroxisomes (a cellular organelle) in control cells and those expressing a protein X. For this experiment I've cultured cells from the same batch in two separate dishes and used a transfection reagent to induce the expression of protein X in one of them. After 24 hours these cells were fixed and prepared for fluorescence microscopy. From each condition I then took 7-9 images of cells, and calculated the number of peroxisomes for each individual cells using imaging software. For each condition I've repeated the experiment 2 more times.
My question is: is N=3 or N=21-27 (3 x 7-9)? Is each individual cell a replicate within the experiment or an independent data point?
Of note, despite the seemingly homogeneous nature of cells in culture, the number of organelles between two cells in the same conditions can vary greatly. Additionally, cell transfection results in different levels of protein expression, creating variability between cells.
From looking at statistical information I would point to N=3, but yet the majority of the articles that I've checked that use this sort of analysis go for N=21-27, or don't mention it. I would like to compare this data using a mean +/- SEM and calculate a two-tailed unpaired t-test.