Welcome to the research world! - Things I Do in The Lab

2. Preparing buffers

Many experiments for growing or handling cells, or initiating a biochemical reaction for certain assays require buffer. The most common buffer that I make is PBS (phosphate buffer saline), which is needed for cell culture at the time harvest to wash away the serum in the media that otherwise inactivate trypsin. PBS is also needed as a solvent for many reagents like dyes, antibodies, and it is a good solution to protect cell pellet or the proteins on blotted membrane, and to maintain the pH. Other buffers such RIPA and loading buffers are quite often used in protein extraction and western blot (see below). I have a long list of types of buffers that one possibly need when working in biological research. Check out my buffer library here to help you make your own!

3. Cell-based assays

I perform many kinds of assays from my cells, such as viability or proliferation, metabolic activity, even the most intricate one, the luciferase assay. Most of these assays have similar working principle, in which reagents could fluorescently label parts of cells (like DNA, membrane, mitochondria) and the quantified fluorescence signal is basically the function of the abundance or activity of those cellular parts. These are called the fluorescence -based assays and usually read by using fluorometer. Measurement of fluorescence labelled molecule to see changes in molecules are also allowed in fluoresence spectroscopy. Range of fluorescence labels are also detected by range of optic filter that excite and emit these fluorescence according to the wavelengths.

There are also assays that rely on enzymatic reactions performed by the cells in which cells may convert reagent A into reagent B, giving some kind of color changes. The resulting colour intensity or the absorbance of the colour measured using filter at certain wavelength. Check out more on various assays that I do here!

4. Microscopy imaging

Picture a large screen computer connected to a heavy laser emitting machine in a dark room! Yeah, that sounds like where I would be on Wednesday afternoon! I do a lot of cell imaging using confocal microscopy. In my current project, I aim to visualize structural changes in the cytoskeleton (actin and microtubules) of stem cells when they are exposed to changes in mechanical environment. When I was working as a research assistant, I also had the chance to confocal image neuroblatoma cells, to see the the effect of certain gene on the microtubules structure. I use confocal because of its resolution and the large focal depth which makes it possible to get the 3D image of cells or tissues as thick as 500 um . Of course, this technology keeps advancing to a system with higher resolution, faster laser scanning, wider focal distance without losing the definition of the image you want to capture. Confocal imaging can also be coupled with other system such as micro-elastography or magnetic field which serves as quantitative platform for studying cells or tissues' mechanical properties or deliver mechanical stimuli while simultaneously imaging the changes in real-time. 

5. Western blot

I do this one a lot, even on weekly basis, when I was working as a research assistant. The aim of western blot is to separate proteins isolated from cells or tissues, base don their molecular weight. As you introduce different conditions to the cells (e.g. drug treatment or a gene knockdown), you want to see whether there is a change in the expression of a particular protein that can be indicative of an important biological process. Any increase or decrease in the protein expression can also indicate whether the drug treatment works for a certain type of cancer or whether a gene or protein is a major regulator of the expression of other genes or proteins. By figuring this out, you can build a correlation and molecular pathway map, helping you to determine which is an effective target for a therapy!

Western blot is actually my favorite and the most trickier among all, as it is a multistep procedure and each step requires a careful setup. Starting from the protein extraction which has to be done at the right temperature to prevent their degradation, then their exact quantification, and storage. Then, you have to make sure that the gels are prepared at the optimum percentage of acrylamide, depending of the degree of separation you want to achieve. The lower the percentage, the better separation you get especially for proteins of interest that have similar size (bands closer together). While running the gel, the anode and cathode connected to the power pack has to match those on the tank. The level of buffer to fill in the tank, the heating and loading of the protein sample to the gel wells, and the gel handling. In transferring to the nitrocellulose membrane, bubbles can get trapped in between, buffer need to be cold, and so many other traps in western blot that if you are not attentive, you will lose the whole 3 day effort. When everything runs smoothly, you will get beautiful bands of proteins with the different level of thickness and intensities (possible quantification) that might be in line with your hypothesis.

6. Polymerase Chain Reaction (PCR)

My other favorite task is measuring gene expression. Yes, quantitative PCR (qPCR) or real-time PCR (RT-PCR) is one way to do it! Please don't be confused as the two are the same!  Prior to performing qPCR, a researcher would usually use cDNA, complementary DNA that is synthesized form the RNA, in a reverse transcription reaction. Thus, it is also called the reverse transcription polymerase chain reaction! 

I do this quite often in my previous job, where I was interested in measuring the mRNA level of certain gene due to various procedures of turning on and off some responsible genes, transiently or permanently. In my current project, I quantify genes in stem cells that are indicative of their capacity to develop into a target tissue. It is a very powerful tool to get insight on what is happening at the gene level and how gene expression can be changed by many factors (chemical, mechanical) even at later stage in development or life, proving that all life is adaptive! As Dobzhansky says, "Nothing in biology makes sense, only in the light of evolution"!!!

7. Flow cytometry (FCM)

Just like the name says, it literally means flowing cell or the study of cells through their flows. So, this procedure is performed to examine the physical and chemical structure of cells or particles. Running cell samples on flow cytometry will help sort cells based on size, separate singlet or doublet, and most importantly based on the interested physical or biochemical characteristics. For example, in my current project I am isolating stem cells from patients tissue samples. Surely, within the block of tissue I will get heterogenous population of cells, or mixture of stem cells, fibroblasts, etc. In order to isolate only the population of stem cells which are known to express unique surface antigens, I incubate my mixture of cells with the fluorescence labelled antibodies that match these antigens and run the cells through flow cytometry. This process will not only give me a pure stem cells culture but also some quantitative insights of how much stem cells can be obtained from different tissues. 

8. Cryopreservation

Cryopreservation means storing or preserving samples (cells, tissue, etc), for future use to maintain their structural integrity and biological functions. Short term storage is usually done in -80C , and cells or most tissues can last up to 1 year in this freezer. For long term storage (up to >10 years), preservation need to be done in vapor phase, or liquid nitrogen tank, at -196 C. I preserve my cells that are still in early culture phase (not passaged/expanded too many times) so I can use them for repeats of experiments. Cryopreserving cells involves harvesting cells and then transferring them into a freezing medium that contains cryoprotectant such as dimethyl sulfoxide (DMSO). Soon after this step, cells in cryovials need to be stored in -80C for at least few hours before being transferred into the vapor phase tank. When you need more cells later, you can simply take a vial and thaw this to revive the cell back in the culture medium. When isolating tissues, it is also best to snap freeze them (directly place in liquid nitrogen) and then transfer to the vapor phase tank.

That is all of the things I regularly do in the lab. Make sure to check out the detailed protocols of each experiment in my other linked posts. I hope these would be helpful !