Part 2: Simple tips and procedures for staining your cells
Updated: Feb 24
Quick tips as you stain (I bring these up because I have made these mistakes in the past!):
-If you’re staining whole blood, make sure you have complete RBC lysis and you wash your cells thoroughly. If you don’t, you will have too much debris and the data acquisition will be messy and likely not usable.
- Check your antibodies in advance to make sure you have enough to stain and that you can stain the panels you want.
- Prepare your buffers and tubes a day beforehand. It makes the process a lot easier.
- Titer your antibodies and check them before using for a big, important experiment.
So now you have some cells to stain. What’s the best way to do it? This blog is designed to be a “cheat sheet” for beginners to flow cytometry.
1. Prepare (A) tubes/plates, (B) buffers, and (C) antibody cocktails.
A. Everyone labels their tubes differently. I have found it most easy to make a numerical list with what is in each tube. If I’m staining in a 96 well plate, I will use a sheet with all the wells and label what is in each one of them, as well as the corresponding tube number. It might be overkill, but then you can always go back and double check what you did if the data doesn’t look the way you were expecting.
B. In this part, there are two, potentially three buffers that you will need for staining.
i. Flow staining buffer: 3-5% FBS, 0.1% sodium azide in 1X PBS
This will be using during the staining and washing period
ii. 1X PBS
You’ll need this for your flow staining buffer, as well as during your live/dead stain.
iii. 2% formaldehyde in 1X PBS
You’ll use this to fix your cells if you are not planning to acquire immediately after staining
C. When staining multiple samples, it is important to be consistent in the antibodies that you are adding. For example, if you find you need to add 0.5 ug of antibody (1μl) to 10 different tubes you are staining, it is much better (accurate) to make a 1 mL stock solution of a 1:100 dilution (add 10 ul antibody to 1 mL flow staining buffer) of antibody and to add 100 ul, than to add 1 ul neat* to each tube individually. By taking from the stock, you’re ensuring that every tube is getting the same amount of antibody. Depending on the panel you’re working with, you can make different types of cocktails and dilutions. An example of this is found further down in the blog.
*neat: When I started flow, I didn’t know what this word meant. But it simply means undiluted and taken straight from the tube.
2. Put your cells in your tubes/plate
Typically, when staining it’s best to stain between 0.3 – 1 x 106 cells. For example, if I am planning to stain 300,000 cell, I will make a cell stock of 3 x 106 cells/mL and add 100 ul to each tube/well I am planning to stain.
3. Live/dead staining
Before you stain with your antibodies, you will want to do a live/dead stain. Dead cells can pick up antibodies and give a false positive. There are a lot of live/dead stains that you can use. You will need to determine which one will work best for you and the colors you are planning to use in your panels. There are a lot of live/dead stains that are excited by ultraviolet wavelengths and won’t overlap with common fluorophores such as PE, FITC, and APC.
When you perform your live/dead staining, you will want to do it in normal 1X PBS without FBS. The dyes used for live/dead staining could be consumed by amine groups within proteins found within the FBS, and not give you an accurate measure of the dead cells you have. Make sure to wash your cells in PBS, and also perform the live/dead stain in PBS. Once you start staining, you can transfer your cells back to flow staining buffer with FBS and this won’t impact the staining.
4. Antibody labeling your cells
Now that you have your cells in their plates, and stained for live/dead, you can start labeling with antibodies! For simplicity, I will only talk about extracellular staining here-intracellular staining will be for a different day.
One of the first points earlier in this blog was that you should titer your antibodies before performing a big experiment. Typically you’ll use 0.1-1 ug of antibodies for 0.3 -1 x106 cells, which usually equates to 0.5-1 μl per sample. This will of course vary depending on the antibodies that you’re using.
I have found it easiest to make cocktails of antibodies and adding them to your cells. I think this is best described as an example, so see below.
A. Antibodies to be used in panel with titered amount shown for 0.3 x 106 cells
CD45 – 0.5 ug (1μl)
CD3 – 0.2 ug (1μl)
CD79 – 0.25 ug (0.5μl)
B. You have a total of 10 samples you want to stain
For this example, we will make a dilution of the antibodies such that you will add 100 ul antibody cocktail to your cells.
*It’s typically good practice to prepare a little bit extra to take into account any pipette inaccuracies; so if you have 10 samples to stain, prepare enough to stain for 11.
CD45: 1μl x 11 = 11 μl
CD3: 1μl x 11= 11 μl
CD79: 0.5μl = 5.5 μl
Flow staining buffer: 1072.5 μl
Total cocktail mixture : 11 μl CD45 + 11 μl CD3 + 5.5 μl CD79 + 1072.5 μl flow staining buffer = 1100 ul
Add 100 ul of this cocktail to your cells, and mix well.
*You can could alter this to make it such that you add 50 ul of cocktail instead, just by simply adding 522.5 μl flow staining buffer instead. It's a personal preference.
C. Incubate your cells and antibodies
I have seen a lot of variation for antibody incubation. When I first started graduate school, we stained on ice, in the dark for 45 minutes. After talking to other researchers, I heard that others did as little as 5 minutes at room temperature. So I took a happy-medium of both, and I typically incubate for 15-20 minutes in the fridge in the dark. You can find what works well for you.
D. Finish your staining!
After your incubation period, you will need to wash your cells with flow staining buffer. You can wash the cells in 3-20X the volume of the original staining volume. Once you wash your cells, centrifuge to pellet. Remove the supernatant and resuspend in 200-400 ul flow staining buffer. If you are not planning to acquire immediately after staining (>4-6 hours), resuspend cells in 2% formaldehyde in PBS. Keep cells in the dark at 4C until you acquire.
Now you’re ready to move to the flow cytometer to acquire your data!