Part 1. Prepping your cells for flow cytometry
Updated: Feb 24, 2020
There are a lot of different ways that you can perform flow cytometry staining. You can use tubes or a 96 well plate, you can incubate in a fridge or on ice, you can incubate your cells and antibodies for 45 minutes or 15 minutes. As you start your staining process, you’ll find what works best for you and optimize along the way. The most important thing is that you stain consistently across all your samples. In this series of blogs, I will describe how to prep your cells for flow, how to stain your cells, compensation, and how to analyze your data. At the end of the series, I’ll provide a basic protocol that can be modified for different uses. Enjoy!
There are lots of different cells you could be working with. Below I outline some ways to handle each of them:
Blood: You can stain whole blood- typically 25-50 ul per staining assay or you can isolate and stain PBMCs (peripheral blood mononuclear cells). If you are looking for rare cells in a PBMC population (consisting of T, B, NK, and myeloid cells), you may want to stain PBMCs instead of whole blood. If you are phenotyping animals (i.e. SCID status), staining whole blood would be sufficient. If you can stain whole blood for your assay- it will save a lot of time.
Tissues: As with a lot of experiments, you may need to stain different organs, lymphoid tissues, or tumors. I have experienced two ways of working with tissues. Both involve initially mincing the tissues into small pieces with small scissors in a petri dish with media.
In one method, I have incubated the minced tissue in a digestion mixture of HBSS (Hanks Balanced Salt Solution) with 300-500 mg/L of collagenase, 3% FBS, and 2mM HEPES, called d-HBSS for simplicity. I would incubate tissues in the d-HBSS at 37 C with rapid vortexing every 15 minutes for 45-60 minutes. After the incubation is over, I pour the suspension over a 100 um cell strainer.
In the second method, I have simply smashed the tissue over a 100 um cell strainer.
I have not done a direct comparison on how this impacts the percentage of dead cells, but less physical manipulation will likely be better for the cells. There are also instruments available to automate this entire process, but they are a bit pricey and won’t be discussed here.
After passing through the cell strainer, you’ll need to wash your cells in a media of your choice. If you're planning to perform functional assays, you’ll want to wash in a serum containing media. Additionally, if you are going to leave the cells in suspension for an extended period of time, it is best to resuspend in a serum containing media. If you plan to just stain the cells right away, HBSS or PBS would suffice for washing.
One tip that can make cell isolation from tissue a little easier is to add DNAase. After smashing your tissues, cells die and release DNA and the cell pellets can become “sticky” and difficult to handle. Adding DNAase can prevent the stickiness.
Sometimes you may need to characterize different markers expressed by cell lines. If you are using adherent cell lines, you will need to trypsinize them to remove them from the plate. Trypsin cleaves C-terminal to arginine and lysine residues; and therefore could potentially cleave your marker of interest. What I have typically done in the past is wait to stain my cells 4-6 hours after trypsinization. If you are using non-adherent cells, you should be good to go. Prior to staining, you can wash the cells in FACS buffer (listed below).
Getting your cells ready to stain
To stain, you’ll either need 12 x 75 mm polystyrene tubes or a 96 well plate. Make sure to be aware of the type of flow cytometer you’ll be using, some are compatible with tubes, while others are compatible with plates. I have also used 1.5 mL microcentrifuge tubes if I am staining very few samples- the centrifuging time is much quicker in a small centrifuge than a larger one. After staining I move the stained cells to the appropriate container for data acquisition.
You should use a staining buffer that contains 3-5% FBS and 0.1% sodium azide (FACS buffer). The FBS (fetal bovine serum) acts to block non-specific antibody binding to your cells, and sodium azide acts as a preservative of the mixed buffer.
I typically stain ~300,000 cells in a 200 ul volume, containing at least 100 ul of FACS buffer when I put the first set of antibodies on. Again, this can vary lab to lab and person to person based on different preferences.
In the next blog, I will describe some tips for setting up flow panels, choosing colors, and how to stain your cells!