The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a widely used colorimetric assay for measuring cell viability and proliferation in vitro. This assay relies on the conversion of MTT into a purple formazan dye by the mitochondrial enzyme, succinate dehydrogenase, in viable cells. The amount of formazan produced is proportional to the number of viable cells, which can be quantified by measuring the absorbance of the dye at a specific wavelength. The MTT assay is a simple, cheap, reliable, and sensitive technique that can be adapted to a wide range of experimental conditions and cell types. It is widely used in drug discovery, toxicology, and basic research to assess the cytotoxicity of compounds, evaluate cell growth and viability, and screen for potential anticancer agents. The MTT assay is also used in combination with other assays and techniques to gain a better understanding of cellular processes and signaling pathways.
Knowing how to perform an MTT assay is a powerful tool. In this tutorial, we will walk you through the theory, practice, and analysis of this assay. In addition, we showcase reagents necessary for the experiment along with downloadable protocols, planning, and analysis templates. Finally, we wrap it up with some real-world examples!
As discussed above, MTT assays are great for looking at cell viability and proliferation. In this brief video, we go over how these assays work and why they work. It's important to understand these things because they directly inform the limitations of this assay. The key takeaway here is that MTT assays are actually measuring metabolism and we estimate viability and proliferation based on that.
Now that we understand the theory, let's talk about how to perform this assay. Below is a list of materials you will need.
Let's go through some worked-out examples of different ways to set up this assay! We'll discuss a basic dose-response assay, and then further show the exact experimental setup, and finally discuss ways the setup can be expanded to accommodate multiple cell lines, drugs, etc.
Antibodies are essential tools in biology and biomedical research, with a wide range of applications across different fields. These specialized proteins are produced by the immune system in response to the presence of foreign molecules, such as viruses, bacteria, or cancer cells. Once produced, antibodies can specifically bind to their target molecules, allowing researchers to detect, isolate, or even neutralize them. Antibodies have revolutionized many areas of biology, from basic research to drug discovery and clinical diagnostics.
In this tutorial, we will explore the basics of how antibodies are used and how they work. We will discuss the structure and function of antibodies and how they can be used in different experimental settings. Let's dive in!
Antibodies have numerous applications. The video above describes the general principles of using antibodies for any of the relevant applications. Below are some of the things they can be used for.
In summary, antibodies have become essential tools in biomedical research, with a wide range of applications across different fields. Their specificity, versatility, and ability to bind to specific molecules make them invaluable for studying biological processes, developing new diagnostic and therapeutic approaches, and advancing our understanding of human health and disease.
Understanding the principles described in the video will allow you to easily use antibodies across a range of applications, including western blotting, immunostaining, flow cytometry, live cell imaging, and much more.
Becoming a doctor is a long and challenging journey that requires years of preparation and hard work. For students interested in pursuing a career in medicine, the pre-med timeline can be particularly daunting. From selecting the right courses to preparing for the MCAT and navigating the medical school application process, there are many steps to take and decisions to make along the way.
In this tutorial, we will provide an overview of the pre-med timeline and the steps that aspiring doctors typically take before applying to medical school. We will cover each stage of the process, from the early years of college to medical school graduation and beyond. Whether you are just starting out on your pre-med journey or are already well on your way, this post will provide valuable insights and advice to help you navigate the path ahead.
The overall picture to keep in mind is that you have four years (or perhaps five, with a gap year) to get your application as ready as possible for medical school. The last year is typically not included in the application, since you will submit over a year before your intended start date, so that should be factored into your planning. In the beginning, you should focus on figuring out if a career in medicine is right for you. Use your classes, activities, experiences, mentors, etc to really explore and decide. After that, the next two years should be spent building up the components of your application. Traditionally, these components are thought of as (1) good grades and scores (2) research (3) clinical activities and volunteering to show exposure to and interest in medicine (4) a clear interest in something within or beyond medicine that a number of your activities, classes, and experiences relate to (5) evidence of leadership and commitment throughout the above. The last year will be spent on the application process, which starts in May of your junior year (for people going straight through) or May of your senior year (for people taking one gap year).
Disclaimer: There are, of course, deviations to this timeline and many different paths to medicine. Perhaps you take multiple gap years, or perhaps you decide in your last year of college and do a post-bac. Either way, if you've decided medicine is for you, don't be discouraged. However, this specific tutorial focuses on a traditional timeline.
If you are taking gap years, move all application-related components in the timeline above (AMCAS, essays, recommendations, etc.) the appropriate number of years out. Use your gap years to deepen your application - pursue meaningful research, work in a hospital, gain medical exposure by being an EMT or a scribe, or do a post-bac if you need to complete pre-med requirements. Talk regularly with advisors at your school or mentors you have identified throughout the process to determine when you are ready to apply.
Undergraduate research is often a key component of your medical school application. If you want to go to academic medicine-focused programs, having a strong research background is one of the things that is closely examined in the process. It's useful to spend your first year exploring so you can choose a lab that focuses on something you're excited about. Ideally, you would to stay in this lab for the next four years and develop a strong relationship with the PI and develop a publication record. If you do switch labs, switch early, find a good fit, and then work to develop that longevity. Finally, if you are doing basic science in a lab that doesn't publish as often, it may be good to supplement with additional faster-moving clinical projects to develop relationships with additional mentors and be able to show publications when you apply.
This website is devoted to helping you develop your research skills and walk into the lab prepared so take advantage of all the tutorials out there. We also have additional tutorials on choosing mentors, identifying projects, etc. so keep an eye out for those.
Sources: The Stanford (Unofficial) Pre-Med Handbook, AAMC Guidelines
Tissue culture is a powerful technique used in modern biology to grow cells, tissues, and organoids in a controlled laboratory environment. It is a complex and intricate process that requires precise techniques and attention to detail. If you are new to tissue culture, it can seem daunting and overwhelming at first. However, with the right tools, techniques, and guidance, it is a skill that can be mastered with practice.
In this tutorial, we will provide you with a step-by-step guide on how to perform tissue culture, specifically focusing on the culturing of cells. However, these techniques can be expanded and applied to tissues and organoids as well. We will cover everything from setting up your space, selecting the appropriate materials and equipment, to preparing cells, and maintaining cultures. By the end of this post, you will have a solid understanding of the tissue culture process, and the skills needed to successfully perform tissue culture experiments in your laboratory. So, let's get started!
The first and most important rule of tissue culture is that everything must remain sterile at all times. Make sure you fully understand how to maintain sterility while working. If your lab regularly cultures cells, you should have an appropriate tissue culture hood, and often a separate tissue culture room, that contains hoods and the cell incubators. This is because it is incredibly easy for cells to get contaminated, and once bacteria or other organisms settle into your cells, the culture is lost and you must start over. As you can imagine, if you have special lines or specific cell types that are hard to culture or obtain, this will set you back massively in your work. There are some basic rules to help you maintain sterility throughout each step of your work.
As you start and get your reagents together for TC, remember to always wear clean gloves when you reach into the hood or the incubators. Don't ever touch anything inside the hoods, incubators, or anything that is marked as sterile with your bare hands. Touching or opening things outside will contaminate them and make them unusable. Before putting your gloved hands into the hood or the incubator, make sure that you spray them down with 70% ethanol to ensure sterility and cleanliness.
Anything and everything that goes in the hood must be sterile. That means that any reagents used inside the hood should NEVER be opened outside the hood. They should be purchased sterile (or sterilized in your lab by autoclaving). Before bringing them into the hood, they should be sprayed down with ethanol. Before taking them out of the hood, they should be tightly closed. They should be stored in a defined location where only sterile items are stored and there should be no mixing between sterile and non-sterile items. (Do pay attention to where they should be stored though - different items may need to be at 4 degrees, -20 degrees, or room temperature). The same applies to other items - pipettes, multi-channels, etc. - they should be delineated as sterile for the hood, be autoclaved, and then only opened inside the hood.
When you work in the hood, make sure that all your items remain sterile as you work. Do not ever open cells outside the hood. Cells go straight from the incubator to the hood and are only opened briefly inside the hood as needed to work. For reagents, maintaining sterility means that anything that touches the inside of your cells or reagent bottles must never touch your hands or any other surface in the hood. (For example, if you have a pipette tip that you are working with, make sure it never touches the outside of any bottles, your hands, or anything except the reagent it is meant for and the cells). Close or cap items as soon as you are done with them. In general, if something touches you or if you are unsure, throw it out and use a fresh tip. Use fresh tips for every reagent and every cell type to avoid contaminating your reagents and your cells with other materials.
Once you are done with the hood, make sure to replace all the items, clean it well with 70% ethanol, and then close it and turn on the UV to fully sterilize it for the next person.
Given the discussion above, let's talk about how to recognize if your cells are contaminated. At first, it can be difficult to tell but you should check your cells daily for signs of contamination. Early signs of contamination include small black dots among your cells or low cell growth and increased detachment. As it progresses, the media will often change color and become progressively cloudy and eventually, the cells will detach and die. As you develop experience, you may also notice that your incubator might start to smell a little funny if there is something in it that is contaminated.
A clean culture should look like this
A contaminated culture may look more like this:
Since the first rule is maintaining sterility and avoiding contamination, the next thing we talk about must be what to do if you do have contamination! The best thing you can do for yourself is to start, if you are new to the process, with a cell line that passages easily, is relatively hardy, and one that your lab has plenty of stock for in case you do have contamination. Examples of these are 293T cells or HeLa cells, which most labs have in abundant quantities. It is also a good practice to separate your media and reagents from your labmates and to keep your cells in a specific part of the incubator so that you can isolate which reagents may be affected if there is contamination.
Once you are comfortable working with those cell lines and are confident that you can work without getting contamination, you can progress to more difficult cell lines. It is still always a good idea to keep your work materials separated from your lab mates, in case there are any issues.
If you get contamination, the best thing to do is start over. The contaminated plates should be soaked in bleach and then dumped. You should obtain fresh reagents you are sure are sterile, clean out your incubator, sterilize it if possible, and then start afresh. If it is not possible to throw away the potentially contaminated reagents (expenses, etc.), another technique is to plate just the media overnight and see if it grows anything. If it does, you know it is the source of contamination, but if not, perhaps you can try again with the same reagents. If you truly are using a very precious cell line or resource, you may try treating it with antibiotics, frequent media changes, and frequent washes to salvage the line. However, this is not a permanent solution if you have significant contamination.
To ensure that you have a fallback, anytime you obtain a cell line or create a line (with a knockout, transformation, or other modification), you should FIRST expand and freeze stock of the line. Freezing protocols are included below. Freezing the lines ensures that you will always have stock that you can start afresh with if you should need it. We would recommend freezing at least 5-10 vials if possible before proceeding with any major experiments.
Now that we've talked about avoiding contamination, let's briefly discuss the broader assessment and maintenance of your cell lines. The goal of tissue culture is to maintain and propagate cells for experiments. If your lab depends heavily on cell models, chances are you will always have an incubator full of cells and at least an hour or two of your day will be spent maintaining those cells.
Part of that time, each day, should be spent looking at the cells and assessing for contamination and confluency, and then maintaining the cells appropriately based on what you find. The important things to do each day are the following
The list above will determine your cell culture maintenance for the day. The contaminated cells should be disposed of to avoid spreading to your other lines. Replate them from frozen if they are required for experiments. The confluent cells should be passaged out to new plates so they are less dense and have room to continue to grow and remain healthy. Cells that haven't had media changes should have fresh media to encourage growth. Finally, cells that are required for experiments should be plated out for those experiments assuming they are healthy and relatively confluent, along with additional maintenance plates so you don't lose the line. If the cells are not ready enough that you could plate your experiment and maintain extra cells, they should be given a few more days to continue growing.
In this last section, I want to briefly discuss how I think about the steps involved in TC. To me, there are four major steps:
We discussed the first step above. This is essential to make sure you have a good stock of cells at all times to be able to produce data.
The second step typically involves washing your cells, removing the media, and then adding in some sort of enzymatic detachment solution (trypsin, EDTA, etc.). This will detach the cells from the plate and allow you to remove them so that they can be transferred to a tube.
The second step, once the cells are detached, is to separate them from the old media. This is usually done with centrifugation in 15mL or 50mL tubes. Since the cells are heavier than the media, they will settle to the bottom when spun. This will allow you to then remove the old media and maintain the cell pellet. It is important here to not lose your pellet when removing the media.
Once the pellet is isolated, you can do anything you want! Step 4 is our decision point. We can either decide to freeze the cells here, using a special freezing media. Or we can plate the cells for an experiment (with additional maintenance plates) which will require counting and then plating cells in appropriate dishes dedicated to our experiment. Or we can simply re-plate the cells into fresh plates at a lower density. When you do this, remember that you do not have to re-plate all the cells - just plate what you think you will need and either freeze or dispose of the rest.
Here are the materials you will need to perform tissue culture.
Tissue culture protocols vary widely based on the type of cell, tissue, or organoid being cultured and the specific media and treatment requirements. However, here is a basic protocol that can be adjusted to your needs.
It is important to follow proper sterile techniques throughout the cell culture process to prevent contamination and maintain healthy cell growth. Additionally, specific cell types may have unique requirements, such as the addition of specific supplements or a different media composition, so it is important to consult the literature or manufacturer's guidelines for the specific cell type being cultured.
Here is a listing of useful resources to assist you:
This is a cell counter sheet that can calculate the total cell number per mL in your sample, and help calculate specific amounts of cells for specific applications.