Cell Viability Assay - Day 6
It's day 6 and our cells have been living in our drug Metformin for 3 days. Today we will start Part 1 of 2 of our Crystal Violet Experiment.
Crystal violet ONLY stains cells that are attached to a culture plate by dyes proteins and DNA within the attached cells. So we want to stain our cells and see how many are "alive" or left on the plate.
Part 1: Staining with Crystal Violet
First off I want to apologise for the lack of images. Crystal violet is messy and I can only take pictures when I'm not wearing a lab glove. So I choose safety over pretty pictures (Nahal would be proud).
The first step with any experiment (as I've shown before) is preparation. I like to get all my reagents, pipette tips etc. ready before I start (as seen in picture 1).
The next step is to get rid off the media from the plates into a sink. We then wash the well (like we did when making our cell suspension) with PBS twice, about 200ul. The PBS is removed each time into the sink (by a highly scientific technique of dumping it out in a chucking motion). 80ul of crystal violet is added into each well (picture 2) and left on a rocker for at least 20 minutes. This spreads the dye evenly around the wells and makes sure the cells that are attached take up the dye.
Part 2: Removing the Crystal Violet
THIS is when it gets messy. This dye stains everything and is difficult to wash off so we first get as much of it out of the well as possible into a specific container in the fume hood (picture 3). We then wash off the excess. We have some very technical equipment here: a bucket and a sink (picture 4). We fill the bucket with cold water and dump the plate into the water. This fills the wells with water and then we chuck it into the sink. You can understand why this gets messy and turns the sink purple (a little ethanol gets that right off). You keep doing this until the water coming out of the plate is clear. This means all the excess is removed and the purple that is left is only absorbed by attached cells.
Part 3: Drying the Plate
It's important to let the plate dry for a few hours or preferably overnight. This allows any water to evaporate. We will be measuring how much crystal violet is in each well tomorrow and we don't want the water to mess up our results. As you can hopefully see in picture 5 and 6, some of the wells have a lot of purple dye and some have barely any.
And that's it for today. I know it is very slow but this really is what science is. When you do experiments the majority of the time you do a little bit one day, a little bit another and you end up waiting for hours and hours for something to happen. Which is why it can be so frustrating when it goes wrong. This simple experiment takes 7 days to complete, imagine the time spent doing more complicated experiments.
So that's it for today. Come back tomorrow to finish the last part of our experiment. We will read the absorbance (I will explain tomorrow) of our crystal violet staining and then analyse our data.
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My name is Caitriona and I am a PhD student at Imperial College London, UK.