Solutions are a routine part of lab life. They are used every day in the lab for a variety of applications, from the stock solutions that are used in almost everyone's experiments, to solutions created specifically for your experiments and maintained as a short-term stock, to solutions mixed as part of experiments for one-time use. They can also include master mixes of multiple reagents created to develop more even plating of reagents and control experimental conditions. There are many different ways that these solutions are referred to and for a beginner in the lab, it can be incredibly confusing to hear these terms thrown around. Don't be overwhelmed! The solutions are simple to make and you will quickly learn to make them. However, make sure you understand how to do it because messing up your dilutions is an easy way to waste expensive reagents and interfere with good experimental results. Here, we'll go over the common terminology around dilutions and we'll discuss how to mix standard solutions.
This refers to the dilutions such as 1x, 5x, 10x, 20x, 100x, and so on. You can, of course, use any number but these are the most common. This description is used for stock solutions that are routinely used in the lab and that are required in large quantities. Keeping a small bottle of a 20x or 100x solution allows the lab to easily generate large quantities of 1x solution as needed. You can also do this for yourself if there is a reagent you use routinely. You make a 20x stock and dilute it out whenever you run out of your 1x stock to expedite the process.
This description means that the concentrated stock solution is 5, 10, or 20 times stronger than what you need it to be. You need to dilute these solutions down to 1x which is the concentration that you actually need. Using extremely concentrated solutions can destroy your cells or tissues, so ensure you dilute correctly before using.
To dilute these solutions think about the concentration as parts of a whole. This means if a solution is 5x and you need to get it down to 1x, you need 4 parts water and 1 part of the stock. That way, it will become 5 times weaker and get you down to 1x. If a solution is 10x, use 9 parts water to 1 part stock. Essentially, if you are using a stock, use 1 part of the stock and then add in all the remaining parts as water until you reach the final number for the stock.
For example, let's say we have a 10x PBS stock and we want to make 1000mL of 1x PBS. To do this, we first need to divide 1000 mL into 10 parts, giving us 100 mL per part. Then, we will add 9 parts water and 1 part stock. This means we will add 900 mL water and 100 mL of the 10x stock to make 1x PBS.
Let's do another example. We have a 20x sodium borate stock and we want to make 100 mL of 1x sodium borate. We start by dividing 100 mL into 20 parts, giving us 5 mL per part. Then, we need 19 parts water and 1 part 20x stock. This means we will add 5mL of the 20x sodium borate and 95 mL of water to achieve our desired 1x concentration. You can see how this would be hugely helpful to a lab. Having just 100 mL of sodium borate 20x stock would allow us to generate 2 liters of sodium borate 1x solution as and when we need it.
The ratio is another very common way of expressing necessary dilutions in labs. This takes two forms - one is with x being a small number - say 1:5 or 1:2 - which works similarly to the dilution discussed in the section above. It means that 1 part out of 5 must be the stock, or 1 part out of 2 must be the stock. That means you would add 1 part stock + 4 parts water or 1 part stock + 1 part water respectively. For example, if I have an MTT stock solution and I want to make 10mL of a 1:10 dilution in PBS, I will take 1 part MTT stock + 9 parts PBS. 10 mL divided into 10 parts is 1mL each, so I will add 1mL of my MTT stock + 9mL of PBS to create the final stock.
The other form of the ratio is when x is a very large number. This is typically used to express antibody dilutions, as antibodies are extremely expensive and extremely potent. To use them, you will typically use dilutions ranging anywhere from 1:50 (at the lowest end) to 1:10,000 (at the highest end). Common dilutions include 1:100, 1:1000, and 1:5000. To calculate these, the 1 is essentially negligible so we don't calculate 4999 parts to the 1 part, we just add 1 part into 5000.
Let's go over this more clearly. Let's say that I am using goat anti-rabbit secondary for an experiment and the recommended dilution is 1:5000. I need 10mL of the 1:5000 antibody solution for my experiment to put my cells in overnight because I have 10 wells that need to stain and each one takes 1mL of antibody solution. So, what does 1:5000 mean? It means that for every 5000 microliters (5mL) that I use, I should add 1 microliter of antibody. We are talking about a tiny tiny amount of antibody relative to the total fluid in the container here. If you find yourself adding a lot, think again and double-check your calculations. Based on the above, for 10mL of antibody solution, I should be able to add 10mL of PBS to a tube and then put 2 MICROliters of antibody in it. I stress this because you do not want to waste antibodies. You will hopefully never find yourself in the position of having accidentally added 20 microliters of antibody to a dilution. However, if you do, you may have just used the entire antibody bottle.
This refers to dilutions such as 1%, 5%, etc. When a dilution is described this way, it's almost always meant as a weight-per-volume dilution, i.e. that you put in a certain weight of a reagent and then dissolve it in a certain volume of diluent. The percentage is always based on a 100 mL volume (which makes sense because it's a percent). For example, if I said we need to make 5% milk in PBS for a western blot, that means 5% weight-per-volume, i.e., 5g of dried milk powder in 100mL of diluent (PBS in this case). You can then scale that formula up or down for the total volume you want to make.
For example, if we wanted to prep 100 mL of milk, we would just measure out 5g of milk and then measure out 100mL of PBS and mix it together. If we wanted to make 50mL, it would be 2.5 g with 50mL of PBS. If we wanted to make 1000mL, it would be 50g in 1000mL of PBS.
For practice, let's work through the following examples.
Let's say we want to make 1000 mL of 1x PBS solution (which is the usable PBS concentration) from a 5x stock in the lab. To do this, we first divide 1000 mL by 5, giving us 200 mL. Therefore, we need to add 200 mL of the 5x stock to 800 mL of water (usually distilled purified water) in order to create a 1x solution.
Let's say we want to make a 1:5 dilution of MTT stock solution in PBS, and we want a total of 50mL of volume. This means 1 part out of 5 should be the MTT stock solution and the rest should be PBS. Therefore, we'll divide 50 mL by 5, giving us 10mL. This means we add 10mL of the MTT stock solution to 40mL of PBS, which will give us a 1:5 dilution.
Now let's say we want to make a 1:1000 dilution of rabbit Notch1 in BSA for an experiment. We want to create a total of 2mL of antibody solution for our plates. To do this, we will first measure out 2mL of BSA and place it into a tube. Next, we'll think about how many microliters 2mL is equivalent to, which is 2000 uL. Now, a 1:1000 dilution means 2 uL of antibody in the 2000 uL of BSA. Therefore, we'll add 2uL of antibody into our 2mL tube of BSA, mix well, and then distribute to our plates.
Finally, let's say we want to mix a 1% solution of milk in PBS for a western blot. We want to make a total of 100 mL so we can cover all our blots. For this, we'll measure out 1g of milk and place it into a 100 mL tube of PBS. We'll mix thoroughly and then be ready to use for all the blots!
As you can see above, the math is easy!! The more you do these calculations, the more automatic it will feel in your mind. Eventually, it will just become second nature. Also, labs usually use the same dilutions over and over again so after a while, you'll just memorize the numbers you need for any given dilution.
To help you out as you get started, here are some online calculators that can help you confirm your numbers.
For more help, check out the protocol section! We have many common lab solutions listed with reliable and easy-to-use recipes. Most standard scientific solutions will have recipes available online through a quick google search as well. Pay particular attention to the storage and longevity of solutions. For things you use often that can be stored, it's helpful to make a large stock once rather than making them over and over again.
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.
