This is to determine whether an extract has an cytotoxic effect on brain cell or specific brain cell,
Depending if your extract is in aqueous form or in ethanol. If aqueou, take your concentrated extract and make a sterile solution (through 0,22 µm millipore filters) in culture medium, then add decreasing volumes of your extract in your cells, but I guess that this extract should not represent more than 5-10% of the total culture medium. Control pH, osmolarity (if possible) and test the toxicity. If your extract is in ethanol add a drop of your extract in the bottom of your culture flask, let it evaporate and then add your cells. You may also add your extract to the culture medium provided that the volume of ethanol represents less than 0.5% of your medium volume. good luck
First you need to see is your extract soluble in medium!!! Make such concentrations that are possible to dissolve in culture medium (needs to be clear-transparent). When you find highest possible concentration that is dissolved start diluting it 2,5,10,50,100 times. After that mix the diluted concentrations with medium containing cells. all best.
Plate you cells in a 96 well plate and culture them until ~80% confluency. Make serial dilutions of your extract in the vehicle then add the same volume of extract to an aliquot (1-2 ml) of media so that you end up with final concentrations of say 200 uM, 20 uM, 2 uM, 200 nM, 20 nM, 2 nM, etc. These concentration will depend on the solubility of your extract in the culture media. Also, you will need to test the effect of the vehicle on your cells. Remove 1/2 the previous media and replace with the media containing your extract. I use one column of the plate for each concentration so that I will can average 6-8 wells (see attached example). After an increment of time, remove the media and perform a cell viability or cell death assay such as Live/Dead. I use a calcein-AM viability assay (but there are many available), normalize the treated columns to the vehicle column, and determine the EC50 of my compound/extract by plotting the normalized viability against the log of the extract concentration. You will have to observe the cells with a microscope to determine when the appropriate time to perform the viability assay is. You could also plate a single concentration across the entire plate and then do the viability assay on one column of cells at increasing time points to determine what is the best time to perform the concentration curve experiment. I do this with primary neurons, SHSY5Y, and C6 cells. Good luck!
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