Cell Viability Assay - Day 1

I am trying a little experiment today. I am aiming to take you as a reader through an actual experiment as I do it. This means taking you through day by day from planning to analysing the data. Hopefully it will give you an idea about (1) how an experiment is run and (2) what you do when it goes wrong. 

Before I start I want to introduce our experimental models. I have three cell lines that we will experiment on:

• Two non-cancerous breast epithelial cell lines - MCF10A and MCF12A.
  
• One cancerous breast epithelial cell line - MCF7. 

DAY 1: Planning

It is important before starting any experiment that you plan what you intend to do. This involves finding out about your experiment (what it is, why you do it, what it does). You then need to work out what reagents and equipment you'll use, how many cells you will need and the timings of plating, treating and completing the experiment. 

Part 1: Crystal Violet Cell Viability Assay
Crystal violet is a common stain used to see the effects of treatments on cell viability (i.e how many cells does the treatment kill over a certain period of time).
In 2D culture, cells that are alive tend to attach to the culture plate. When the cell dies it detaches from the plate and floats in the media. Crystal violet ONLY stains cells that are attached to a culture plate by dyes proteins and DNA within the attached cells. The amount of crystal violet can then be measured and this is used as an indirect way of quantifying cell death/viability.

Part 2: Equipment and Reagents
Buffers/Reagents:

• DMEM/F12 Media: media used to culture MCF10A and MCF12A non-cancerous breast epithelial cell lines
• DMEM media: media used to culture MCF7 cancerous breast epithelial cell line
• EDTA/Trypsin: trypsin - serine protease that allows the cells to detach from the bottom of the flask by degrading peptide bonds. EDTA – chelating agent that forms bonds with Mg2+ and Ca2+
• Metformin hydrochloride: this is the drug I am testing 
• PBS: Phosphate Buffered Saline. Non-toxic solution used as a wash buffer or a dilution buffer. Typically has a pH of 7.4.
• Crystal Violet Reagent: 0.5% crystal violet in 25% methanol. This purple dye only stains cells attached to the plate.
• 10% Acetic Acid: used to detect crystal violet absorbance.

 
Equipment:

• 96-well plate
• 5ml, 10ml and 25ml stripettes
• Automatic pipette boy
• 50ml falcons
• 50ml reservoirs
• p10, p20, p200 and p1000 pipettes and appropriate tips
• p300 multichannel pipette and appropriate tips
• Rocker
• Water Bucket

Part 3: Number of Cells and Timing
To determine how many cells you need for your experiment you need three pieces of information: (1) the doubling time of your cell line (i.e the time it takes for the amount of cells in your flask to double), (2) the time point you wish to investigate and (3) the size of your experimental plate.
For this experiment:

• Doubling Time: approx. 29 hours (the doubling time for each cell line is slightly different but the average across the three is 29 hours. The DT is close enough between the three that it works to use the average).
• Time point: 72 hours or 3 days
• Experimental Plate: 96 well plate

NOTE: weebly doesn't allow me to superscript so whenever you see this "104" please note that is means "10 to the power of 4 or 40,000"

​I know that when my 96-well plate is full there is theoretically 4 x 104 cells. You then use this as your final time point and half the number of cells until you reach your start time (see picture 1). As my cells aren't all doubling at exactly 24 hours, I reduce my end confluency a little to account for this. You can then work out how much media you need (each well takes 0.1ml) and how many cells you need (see picture 2). I will be doing two rows (12 wells in each row) for each cell line (see picture 3) and I will do two plates (I'm doing a similar but different experiment at the same time which I will explain later). So the total number of wells is 48, rounded up to 60 in case of error. Picture 3 shows the plate plan. Experiments are always run in triplicate (sometimes more). This is so we can average the results and reduce technical error (i.e. error by me). We will be treating our cells with increasing concentrations of our drug Metformin, shown on the plan.  

Picture 1: How many cells you need to complete your 72 hour experiment in a 96 well plate.

Picture 2: Calculations to work out how many cells you need and how much media you need.

Picture 3: Plan of the 96-well plate we are conducting our experiment in. This plate plan is duplicated for the other experiment I'm running.

Part 4: Our hypothesis

​It is important to have a hypothesis before we begin. This is a question we are asking which we hope our experiment will answer (either positively or negatively).

Hypothesis: Metformin reduces cell viability of MCF10A, MCF12A and MCF7 cell lines. 


So that's it. Tomorrow we will set up our experiment and I will show you how we finish our calculation from picture 2.