Grad student with a "simple" problem.
Im aiming to test the neuroprotective effects of a compound series against a neuroblastoma cell line, against a know neurotoxin. The general goal is to use a 24 well plate, and test cell viability via MTT.
Im running into a concern with confounding variables, and restricted space. is there an easy way to normalize my results across several plates?
Controls:
cells + MTT
cells + neurotoxin + MTT (negative control)
cells + test compound + MTT
neurotoxin + MTT
test compound + MTT
this leaves 2 trial runs for varying concentration of test compound, which is rather limited.
It would be easier to do my controls on a separate plate, and my experiment w/ a cells+MTT control on a separate plate, and then normalize the data.
Any suggestions? 24 well plate was picked because of differentiation stipulations that need to be addressed before exposures
Hi Kelly
From previous experience with tox assays I would suggest using a 96 well culture plate. In stead of MTT I suggest you use the soluble tox assays such as XTT or WST1. You can then read your plates directly on a plate reader at multiple time points after addition of your test substance.
First check if the neurotoxin & test compound affect XTT/WST1 readings. For this you do the following wells (NB done in absence of cells):
XTT/WST1 only (control)
XTT/WST1 + neurotoxin
XTT/WST1 + test compound.
Take readings at various time points after addition. Calculate the change of absorbance per hour. If the compounds have no effect on absorbance compared to the control you can proceed.
Next exp is toxicity of neurotoxin and test compound.
Grow cells in a 96 well plate. Add you toxin and test samples. Add XTT/WST1. Take readings at various time points after XTT/WST1 addition to cells.
Wells must contain:
Cells plus XTT/WST1
Cell + neurotoxin + XTT/WST1
Cell + test compound + XTT/WST1
Vary concentration of neurotoxin and test compound.
Take readings at times after XTT/WST1 addition. Calculate change in absorbance per hour.
Plot absorbance change per hour (y axis) vs neurotoxin concentration (x-axis). From this calculate IC50.
Do the same for test substance. Hopefully not toxic ie slope is 0.
Next exp will be to test neuroprotective effects of test compound.
Grow cells in a 96 well plate. Add your neurotoxin at IC50 to one half of plate. Add dilutions of test samples. Add XTT/WST1. Take readings at various time points after XTT/WST1 addition to cells.
Left half of plate (control):
Row A: Cells plus XTT/WST1 (100 % viability)
Row B-H: Cell plus test compound at various concentrations plus XTT/WST1
Right half of plate (neuroprotective effects):
Row A: Cells plus neurotoxin at IC50 plus XTT/WST1 (50 % viability)
Row B-H: Cell plus neurotoxin at IC50 plus test compound at various concentrations plus XTT/WST1.
Take readings at times after XTT/WST1 addition. Calculate change in absorbance per hour.
Plot absorbance change per hour (y axis) vs test compound concentrations for the two data sets. Hopefully test compound increase viability of cells (increased absorbance) in presence of toxin. This will indicate neuroprotective effects.
Im currently restricted to a 24 well plate for the experimental model (any tests that require cell exposure).
I would assume that the qualitative information gained from a 96 well "interaction plate" would be applicable to a 24 well "experimental plate". would my assumption be correct?
Would be correct. You can use a 24 well plate. Hopefully you have a plate reader that can be set on various plate modes. Just check the instruction manual of the plate reader. My plate reader can do 6, 12, 24, 48, 96 and 384 well plates. Can be done at press of key on the operation programme.
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