A Day in the Lab with an IBC Researcher

One of the most routine cell culture tasks to keep cells alive is feeding them. To feed them, we exchange the liquid media with fresh media every 2-3 days. The picture shows me using a glass pipet to suction off the old media before adding new media.

In studying responses to drugs or genetic responses to gene ablation, we often perform cell proliferation experiments in plates like these which have 96 little wells, that we add 1000-5000 cells per well, then add drugs at a variety of concentrations to find the optimal doses for more detailed mechanistic studies.

When the drug incubation time has been completed (3+ days usually), the final step in measuring the cell number is to add a chemical called MTT (which we dilute in media) to the cells It is a yellowish powder, that live cells convert to an insoluble purple powder over the course of several hours. Here we are adding the diluted MTT dye then we put the cells back in the incubator for 4 hours to let the live cells make purple! The degree of purple we read later and that tells us how many cells are left over.

At the end of the experiment you see the wells have some purple (looks more dark red because of the media). Here we use the metal device with prongs (we call it an “aspirator” in the lab, but that’s not its formal name) to such up the media. Then we add a chemical mix to solubilize the purple to make a purple

If the cells have spread out to cover 70-80% of the surface of the plate, in order to keep them growing well, we need to detach them and replate in fresh plates at a lower density and/or setup new experiments. If we are just keeping some of them, this is called splitting or passaging them. We detached the cells using an enzyme called trypsin, then we spin down the cells in a conical tube in a big centrifuge to pellet them.

This is the tube after we are finished spinning them – can you see the pellet containing a few million cells? Next we will aspirate (suck off) the media and add some fresh media.

It turns out we were using these cells to setup a new experiment, so we counted them using a small portion of them in this cell counter (that costs ~$10-15K). That way we can start out with equal cell numbers in the control and drug treated plates. We will also plate a small portion of them just to maintain for future experiments in the next 3-4 weeks.

Here we are in the flow cytometry core laboratory reading samples that have been prepared the day before. We have added a red dye that binds to DNA and fluoresces when it is excited by a laser. The purpose of this is to know how much DNA is in each of the cells, which we use to determine what phase of the cell cycle they are in since we are studying cells that arrest the cell cycle. The glass tubes in the carousel contain our samples, and Angela is working on the computer to tell it what samples are in each tube, and setup the protocol for reading. The flow cytometer is behind Angela (sorry!)

This is what the data looks like as it is being read by the flow cytometer. There are 2 peaks, and we care about how big the peaks are, especially differences between samples. The report gives us the percentage of cells with each amount of DNA (1 copy, 1.01-1.99 copies, 2 copies, >2 copies) based on the boundaries we set in the program.