variables

[att159]

hello,
i am conducting an experiment will the following design:
macrophages will be treated with beta amyloid and one of four other substances (testing them for properties of inhibiting beta amyloid tnf-a production). then, macrophage supernatant samples are taken and tested for tnf-a at 3 and 48 hours. the rest of the macrophage medium will be transferred to a neuroblastoma cell line, and cell viability counts of the neuroblastoma cells are taken at 3 and 48 hours. (purpose is to determine effect of beta amyloid induced tnf-a on neuronal apoptosis) then, macrophages are lysed and gene expression is determined using dna microarray.
my question is, when conducting the elisa and microarray do i need to include samples of macrophages treated with only the inhibitors?
i was planning on:
cells with no treatment at all
dmso vehicle control
cells with beta amyloid but no inhibitor
cells with beta amyloid and inhibitor w, concentration 1
cells with beta amyloid and inhibitor w, concentration 2
...
cells with beta amyloid and inhibitor w, concentration 6
cells with beta amyloid and inhibitor x, concentration 1
...
and so on, until cells with beta amyloid and inhibitor z, concentration 6.

originally i did not plan on treating cells only with inhibitors w, x, y, z (no amyloid). is this necessary? i think the microarray expression data could be interesting with just the inhibitors, but i will probably only do it if it scientifically necessary in terms of experimental correctness.

thank you!

[carolinethorn]

Wow this is a very creative series of experiments. I really think they could occupy a Ph.D. student for a whole year working full time on it and it could give some very interesting data and papers out of it.

I think it would be helpful to have cells treated with inhibitor only, it would be more complete. Particularly for the experiments with adding supernatant to the neuroblastoma cells.

I hope you don't think the following is negative but I am going to try and dissuade you from being too ambitious with how much of this you attempt to do at a time. We read scientific papers and often forget to think about how long it took for the authors to assemble all the data needed for a paper - often more than a year. Each figure in the paper may have been more than one experiment that was done with each treatment in triplicate (at least) and then the whole thing would have been done three or more times to be sure what they saw was the same.

If you were my Ph.D. student I would be asking you to start with a preliminary experiment to get the feel of how the cells grow and replicate the finding of how beta amyloid stimulates TNF production. Then tackle each inhibitor in turn. Probably starting with the ELisa experiments and leaving the neuroblastoma ones til later.
How are you going to organise these experiments? What will you do in the first set? Have you already done a preliminary experiment with different concentrations of beta amyloid and negative control? What size wells are you using to grow your cells in and how many wells do you have for each treatment group?

Don't get me wrong, I think its great you are aiming high and tackling such a great project but in order to be sure you are really going to get results you can interpret sometimes you have to do things as a progression in bite sized pieces.
By the way, what inhibitors are you thinking of using?

Best of luck,
Caroline