Cell Viability Assay - Day 3

It's Day 3 and today we will be treating our cells with the drug Metformin. 

Just a bit of background on Metformin. 
Metformin is a Type II Diabetes Mellitus (T2DM) drug that has been used since the 1950s. It is the most commonly prescribed T2DM drug meaning most people who have T2DM will have been prescribed Metformin at some point during the course of their disease. We know that Metformin targets mitochondria. Mitochondria are the batteries in your cell where the energy to run your cell is made. You can have hundreds and hundreds of mitochondria all working to keep the cell ticking along. Interestingly, all your mitochondria come from your mother. In T2DM you develop insulin resistance. Basically insulin (a hormone made in your pancreas) tells cells to take up glucose from the blood after eating. But T2DM patients make their OWN glucose and don't need to listen to insulin. Your pancreas finds this confusing so churns out more and more insulin, thinking "they'll listen to this!" Eventually your pancreas gives up and you have all this unused glucose floating around in your blood. What Metformin does is it stops your cells from making its own glucose. This forces the cells to listen to insulin and start taking up glucose from the blood. But Metformin also interacts with a whole HEAP of other pathways in cancer alone (some of which are in picture 1) and no one knows why or exactly how. Which is where I come in (in part)! 

Picture 1: Metformin Proposed Mechanisms of Action in Cancer. (1) Metformin inhibits mitochondrial respiratory chain I which leads to decreased ATP and increased AMP. (2) Increased AMP leads to increased AMPK, which inhibits mTOR. (3a) Metformin inhibits Cyclin D1, which prevents cells from going into G1/S phase. (3b) Induction of apoptotic intermediates such as cleavage of Caspase-7 and cleavage of PARP1 leads to apoptosis. (4) Metformin induces cancer stem cell death.

So now we know what our drug does, on to the treating. 

Part 1: Planing the treatment
So before we start with the treating we need to know how much we are treating with in mM and convert this to a volume. I've done this part as a picture (picture 2) as it's easier to explain. 
We have our stock concentration of Metformin which is 1M or 1,000mM. We want to have a gradient of 7 concentrations (see picture) so we need to work out what volume of drug we need to have that concentration in each well. We then scale this up to however many wells we need (in our case it's 10).
The calculation is:

                 (WANT/HAVE) x FINAL VOLUME

​This gives you the answer in ml. We want ul so you multiply by 100 to get the volume of drug for 1 well in ul (see table). We want to do this for 10 wells so we multiple the volume of drug again by 10 giving up the volume of drug in ul for 10 wells (see table). We then need to calculate how much media we require to dissolve our drug into. Because we don't want to disturb the cells at this point, we don't remove all 0.1ml of media. We instead remove a 5th of the media (or 20ul) per well. So you multiply 20ul by the number of wells you want to treat giving you 200ul total. We then take the volume of drug away from the total volume of media (like with our plating) leaving you with the volume of media in ul for 10 wells (see table). The cells will still receive the same amount of drug in each well don't worry.  

Picture 2: Treatment plan for Metformin 96-well crystal violet assay

Part 2: Treating Our Cells
Once we've calculated all of the above we then make this up in labelled 1.5ml eppendorf tubes. Again we will use a multichannel pipette to treat the cells (due to the wrist injury) so we transfer our drug + media into labelled reservoirs (see picture 4 - picture from earlier experiment apologies). We add the appropriate drug to the appropriate wells (e.g. 2.5mM to the three 2.5mM wells) and leave in an incubator at 37°C and 5% CO2 for 72 hours or three days. The cells don't need to be retreated because this a short time course experiment. 

And there you have it! We have now plated and treated our cells with our drug. We now wait three days to allow the drug to do its thing. 

Come back on Tuesday 7th March to see how we do the next (and messiest) stage of our experiment: Crystal Violet!