Antibody Basics


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!

Video Overview

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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.

  1. Immunodetection: Antibodies can be used to detect the presence of specific molecules in biological samples, such as proteins, DNA, or RNA. This technique, known as immunodetection, is widely used in research and clinical diagnostics, and can help identify biomarkers of disease or monitor the expression of genes or proteins in different tissues.
  2. Immunoprecipitation: Antibodies can also be used to isolate specific molecules from complex biological mixtures, such as cell lysates or serum. This technique, known as immunoprecipitation, allows researchers to study protein-protein interactions, identify novel protein complexes, or isolate specific proteins for further analysis.
  3. Flow cytometry: Antibodies can be conjugated to fluorescent dyes and used in flow cytometry, a technique that allows researchers to analyze and sort cells based on their surface markers. This approach is widely used in immunology and cancer research, and can help identify specific cell populations or characterize the immune response to different stimuli.
  4. Therapeutic applications: Antibodies can be used as therapeutics to treat a wide range of diseases, including cancer, autoimmune disorders, and infectious diseases. Monoclonal antibodies, which are produced from a single clone of immune cells, are particularly useful in this context, as they can be designed to specifically target disease-causing molecules while minimizing side effects.
  5. Antibody engineering: Advances in antibody engineering have enabled the development of novel antibody-based therapeutics with improved properties, such as increased specificity, affinity, or half-life. These engineered antibodies, also known as biologics, have revolutionized the field of drug development and have led to the development of several blockbuster drugs.

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 Big Picture

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).

This hexagon essentially encompasses the significant components of your application that you would want to show strength in. No single application is strong in all of these factors so don't expect that of yourself. Identify things that you want to focus on and excel in. Try to make sure they naturally build towards one clear goal or interest that can align with why you want to go into medicine.

Year By 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.

Year One
  • Focus on exploring at first. Join clubs and activities that allow exposure to healthcare or other interests.
  • Identify and speak to potential advisors. Discuss course selection and specific experiences or opportunities that exist at your school that may be relevant to your interests within or outside medicine.
  • Attend campus pre-med meetings and make sure you are on email lists to get relevant updates and information
  • Figure out what you might be interested in studying and what kind of research you may be interested in pursuing. Through your classes, research, and volunteering, start to develop relations with faculty, advisors, and mentors.
  • Seek opportunities to volunteer, shadow a doctor, and, if interested, identify research opportunities on campus.
  • Start taking the required portions of the premed core curriculum at your school, geared for freshmen.
  • Towards the end of the year, line up a plan for the summer, ideally focusing on some combination of volunteering and research. 
  • During the summer, get involved in medicine-related activities - consider internships, volunteering, research, and leadership opportunities. Take summer courses through a university if desired or necessary. However, we would not recommend this unless you absolutely need to. Otherwise, it is better to spend the time investing in an experience or opportunity that you can meaningfully gain from.
Year Two
  • Check in with your pre-health advising office about your progress during first year
  • Continue to pursue meaningful clinical experience, medically-related activities, volunteer work, research, and/or leadership roles
  • Continue to develop relationships with faculty, advisors, and mentors.
  • Apply for summer research, internship, or enrichment programs. Ideally, continue or expand on your activities of the previous summer. However, if it wasn't a good fit, this is a good time to switch and invest in something new. 
  • We would recommend a depth of activities over quantity - i.e., if you had a good experience, invest in that experience rather than bouncing around and trying new things because that will allow you to develop long-term relationships and experiences.
  • During the summer, Continue your summer research, internship, and enrichment plans developed during the year. By this summer, if you are planning to apply straight through, you should have been able to identify which activities you really want to invest in. Use this summer and the beginning of your third year to really develop those relationships, push forward publications, gain leadership positions, etc. to bolster the upcoming application.
Year Three
  • Identify and pursue leadership opportunities within organizations you have been deeply involved with through college
  • Consider which faculty, advisors, and mentors on your campus you have close relationships with, and whom you can approach to write letters of recommendation for your applications.
  • Continue your participation in meaningful clinical experiences, other medically related activities, volunteer work, research, and/or leadership roles on campus; if possible, consider taking on a more substantial role.
  • Meet with your pre-health advisor to strategize about your application timeline, whether it be for immediately following graduation or after one or more gap years. Discuss your schedule for completing the remaining premedical coursework and other school-specific degree requirements.
  • Identify the best time for you to take the MCAT® exam; visit the MCAT website to find the best options for test dates and locations. Take the exam by the end of this year if you plan to apply straight through.
  • If you are considering a gap year, investigate a meaningful paid or volunteer medically-related experience to complete during that time.
  • Research medical school curricula and joint, dual, and combined-degree programs and start templating a list about where you will apply towards the end of this academic year.
  • Request letters of recommendation and committee letters and start preparing your personal statement and the rest of your application materials.
  • During the summer, continue your involvement with your selected summer activities - paid, volunteer, internship, medically related, research, and leadership experiences.
  • If you are applying to medical school going straight through, this summer is the time to complete and submit AMCAS and secondaries. Admissions are rolling so the earlier you do this the better.
Year Four
  • In the fall through early spring, if you are going straight through, you should be interviewing with schools and doing campus visits this year
  • Continue investing in your activities
  • Toward the end of the year, send letters of interest (with key updates) as needed
  • Say yes to the school of your choice and plan to start medical school in the fall!
  • Enjoy this summer if you are going straight through. Remember, it will be one of your last real vacations for a long time. Travel, relax, and do something you are really excited about.
Gap Years

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 HandbookAAMC 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!

Key Concepts

Maintaining Sterility

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

Pulmonary Cell Culture - PromoCell
Note the clean cells, uniform shapes, and lack of black dots or oddly shaped cells.
There are also no floaters in the media that are visible
Image source: Wikipedia

A contaminated culture may look more like this:

What is the reason for my cell-line contamination at day 3?
Note the floaters, the cell death, and the cloudy forms of bacteria covering the culture.
Image Source: ResearchGate
Maintenance & Rescue

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.

Assessing Cells

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

  • Check EVERY plate of cells and maintain only what you need for experiments and freezing
  • Identify cells that may be contaminated (if applicable)
  • Identify cells that are 80-100% confluent and require passaging
  • Identify cells that are not confluent but have not had media changes in 2-3 days
  • Identify cells that are required for experiments and need to be plated

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.

Basic Steps

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:

  1. Assess cells and decide your plan
  2. Detach cells from the current plate
  3. Separate the cells from the media
  4. Isolate the cells and re-plate them for an appropriate application (maintenance vs experiment)

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.

  1. Prepare the cell culture media: Depending on the cell type, the media composition can vary. Generally, it contains a basal medium, such as DMEM or RPMI, supplemented with serum, antibiotics, and growth factors if needed.
  2. Prepare the cell culture vessel: Tissue culture-treated plastic dishes, flasks, or plates are commonly used. Sterilize the vessel and any tools or equipment that will come into contact with the cells.
  3. Seed the cells: Count the cells and add them to the vessel at a specified density, typically between 1,000 and 10,000 cells/cm². Gently rock the vessel to distribute the cells evenly.
  4. Incubate the cells: Place the vessel in a cell culture incubator at 37°C with 5% CO2. Monitor the cells regularly for growth and confluence.
  5. Subculture the cells: Once the cells have reached confluence or the desired level of growth, they can be passaged into a new vessel. This involves detaching the cells from the original vessel using trypsin or another cell detachment reagent and reseeding them into a new vessel at the desired density.
  6. At this point, you should seed some cells for maintenance and plan others for experiments and seed them accordingly.
  7. Replace the media: Depending on the cell type and growth rate, the media may need to be replaced every 1-3 days. Carefully aspirate the old media and replace it with fresh media.
  8. Monitor the cells: Observe the cells regularly for any signs of contamination, abnormal morphology, or growth characteristics.

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.