After a short break, I'm ready to continue with writing about all the fun I have in the lab while doing my research! :)
Now that we have established our own model system (transgenic cell line) which stably expresses our gene of interest, we can continue with downstream experiments which will (hopefully) help us determine the effect of our overexpressed gene in our model system, and potential implications of that overexpression in Multiple Myeloma.
Once you have your model system, one of the first things you want to check is sensitivity of cells overexpressing gene of interest to different drugs, both to those already in use and to new ones.
Image credit: Pixabay.com
One of the best ways to do that is to perform a high-throughput drug screen, meaning that you have to test a large number of different compounds to potentially find several drugs that will specifically target cells with high expression of your gene of interest.
But, what is exactly that large number of drugs you may ask?
Well, couple of thousands, or to be precise - 1902 different compounds in our case!
Wait, that's impossible, a human cannot test all those drugs by hand! Maybe a robot can?
Exactly! But where do we find one?
Luckily, there are special places called screening facilities, where you can find professional and awesome team of people ready to help us investigators with our experiments. They are offering their services of high-throughput screens that can meet a large variety of needs.
One of such facilities is ICCB-Longwood Screening Facility at Harvard Medical School of Boston, and we had a privilege of using their services for our drug screen.
How does all this work?
First, you appoint for a meeting with stuff of screening facility where you describe your experiment in detail. Then you select which compounds you'd like to test in your experiment from their compound libraries. After establishing all important points and performing a test experiment to set up the instruments for your particular experiment/cell line, you're ready to start!
To be able to perform a high-throughput drug screen, first you need to grow your cells in special plates containing large number of wells. For that purpose, 384-well plates are most often used.
Image credit: NIH-NCATS by Daniel Soñé Photography, LLC, CC BY 2.0
Just so you get insight into the size of these very, very small wells - each of 384 wells has maximum volume of 120 microliters (µl), that is - 0.12 milliliter (ml)!
In our experiment, we seeded 30 µl of our cells in each well, for which we used the instrument called Multidrop™ Combi Reagent Dispenser.
Then different compounds were added in each well, 1902 compounds precisely, and the volume of each compound was 33 nanoliters (nl), or 0.000033 ml! Do you have any idea how small that volume is? So small that it's not visible with naked eye!
Of course that a human cannot pipette volumes that small, and that's why there are special robots who perform so called "pin transfer" of compounds. They are transferring nanoliter volumes of your desired compounds from storage wells into your 384-well plates using stainless steel pin arrays. Cool, isn't it? :)
To get a better picture of what I'm talking about, check out this super cool video of pin transfer process I personally made while performing drug screen at ICCB-Longwood Screening Facility - make sure to pay attention to the giant robotic hand grabbing plates and transferring them from the rack to the pin transfer spot!
After the incubation time has passed (24, 48 or 72 hours), you need to perform cell viability assay to determine survival rates of your cells for each compound.
For that, we used luminescence-based assay, during which we quantify the ATP content in cells, meaning that there are metabolically active cells present. During the luciferase assay, in the presence of ATP, mono-oxygenation of luciferin is catalysed by firefly luciferase, and luminescence signal is emitted.
Image credit: Wikimedia Commons, public domain
In the end, obtained luminescence signal is detected by plate readers, the raw data is analyzed and survival rates are calculated.
Raw luminescence data - this is what you get as an output from a plate reader
After you had a tremendous amount of fun with your raw data, and finished analyzing the effect of 1902 compounds on your cells, next what you should do is select candidates for cherry pick screen.
This basically means that you have to select the most active compounds, the ones that showed differential effect on survival of your cells - for example, the ones that preferentially killed cells expressing your gene of interest.
We selected 47 active compounds, and proceeded to cherry pick screen, which I had to perform myself (no robots this time!). We switched to 96-well plate format, because now we have less compounds to test and also because they are possible to pipette by hand.
Look at all this plate art made by me - decreases your chances of making an error significantly!
The analysis of our cherry pick screen data is still in progress, but basically, we need to select a few compounds for further testing (other cell lines, animals, etc.) I will let you know how that party went! :)
Until then, relax and keep steemSTEM! ;)
Literature
A luciferase based viability assay for ATP detection in 384-well format
