Flow cytometry training is crucial to make informed decisions and designing your protocols to get good data. Do some background reading on key topics like fluorescence and hydrodynamic focusing. Know why you want to use the flow cytometer and whether your application is general or niche. Participate in practical training to cement your knowledge.
So you’ve got your flow cytometry training booked and are one step closer to that precious data.
In our experience, the students who hit the ground running with their flow cytometry experiments are those that fully engage with the training program. Those who don’t—well, let’s just say their first few flow attempts aren’t much fun.
Typically, flow cytometry training will consist of some theory plus a practical session to learn cytometer operation and data acquisition. At our facility, the format is as follows:
- ‘Intro-to-flow’ online course plus some cytometer operation videos.
- Training session 1: (or, ‘how not to break the cytometer!’).
- This is run in groups using beads provided by the facility and covers basic steps such as cytometer startup, changing sheath/waste, priming, setting up an experiment in the software, instrument settings (voltages/gains), compensation, and simple hierarchical gating.
- Training session 2: users bring their samples and set up their specific experiment, applying knowledge and skills from session 1.
- Follow-up support as requested until a user feels confident.
Here’s my advice on how to get the most from your flow cytometry training.
1. Know Why You Want to Use Flow Cytometry
Don’t rock up to training without an idea of what flow cytometry is and how you want to use it in your own research. You’d be surprised how many students turn up because their supervisor told them to, but have no idea what experiments they will perform.
You’ll have better questions, and facility staff will be better able to target the session toward your needs.
- The sample type. Will you work with whole blood/PBMCs, digested tissue, or cultured cells? Your facility will be able to advise you on sample prep and provide tips for sample-specific instrument setup.
- The type of experiment you will perform. Will it be immunophenotyping or a functional assay such as cell cycle or transfection?
- Will you perform a multi-color experiment, or do you have a simple panel (combination of antigens/fluorophores and other fluorescent dyes)? This may influence the type of cytometer you undertake training on and will help facility staff identify what other concepts you’ll need to learn, for example, compensation.
- Will you be building on a protocol already developed in your lab, or do you need to start from scratch? Be aware that designing a new flow cytometry experiment from the start can take 6 months or more.
2. Trust the Process: Do the Pre-work
If your facility asks you to do some prep work in advance of your training—do it! They’ve designed it that way based on years of experience. The intended learning outcomes of the practical session will build on the pre-work. You don’t want to get caught out!
In training session 1, we ask users to demonstrate cytometer operation, such as changing sheath and priming. Activities build on concepts from ‘Intro-to-flow’, for example, gating on single cells.
If there is no pre-work required, do your homework. We recommend that you familiarise yourself with the following concepts:
- Hydrodynamic focusing.
- Forward and side scatter (FSC and SSC) and interpretation of FSC v SSC plots.
- How to interpret excitation and emission spectra.
- How lasers are used to excite fluorophores and filters used to collect emitted fluorescence.
- Pulse processing and how to use area (A), height (H), and width (W) to gate out doublets.
- Spectral overlap and a basic understanding of compensation.
- Hierarchical gating for data analysis.
3. Be Prepared and Be On Time
Core facilities are busy places, and facility staff have tight schedules. If you are late you’ll miss important information or won’t have time to complete your experiment set-up. Make sure you know where to find the facility, and if you must prepare samples, allow plenty of time—it always takes longer than you think! Even better, stain them the night before and fix them.
Find out if your flow cytometry training will use beads provided by the facility or if you are expected to provide samples. We split our training into two sessions. The first is a group session using beads, and the second is bespoke with user samples. This enables the learner to apply their skills from session 1 to set up their actual experiment.
If you are asked to prepare samples, make sure you know:
- A cell staining protocol. Ask your facility if nobody in your lab has performed this.
- What buffer to bring your cells in. This is usually PBS or FACS buffer (PBS plus FBS and EDTA).
- What tubes to bring your cells in (e.g., ‘FACS’ tubes: 5ml round bottom tubes, Eppendorfs, etc.).
- Which controls are necessary to analyze your data, for example:
- Compensation (single color) controls.
- Fluorescence minus one (FMO) controls for gating.
- Unstained control.
- Positive/negative experimental controls.
- Do your samples require fixation?
- How will you transport them safely?
- What if there is a spillage – will you clear it up?
One or two samples plus controls is ideal for training. Something representative of your real experiment, that can be considered a practice. Don’t turn up with 50 tubes or those precious clinical samples. The goal of training is not the experiment itself but to get you confident with setting up the cytometer to acquire high-quality data.
Don’t forget to bring any handouts given by the facility, or a notebook/laptop for taking notes.
4. Go With the Flow
Get stuck in and engage with activities. For our practical flow cytometry training sessions and workshops, we design an activity to deliver each learning outcome. These require student participation with content and peers. Experiential learning, or ‘learning by doing’, leads to a deep understanding in a technical setting. 
For example, we give users a list of fluorophores that will be used in the training experiment and ask them to select the correct laser/filter. This enables us to assess their understanding of this concept, and for you to identify any gaps in your knowledge. It’s not a test. We just want to target further instruction where required.
Ask questions! Facility staff love to talk about flow cytometry—so ask them. They’ll also be asking you questions to check that learning is taking place. Questioning, discussion, and explaining facilitate learning. Remember that there are no stupid questions, flow facility staff were in the same position as you at one point.
In fact, it’s a two-way process. Students often have a perspective we’ve never considered before, and it helps us learn too.
5. Embrace Collaborative Learning
Social constructivism is the theory that knowledge is acquired when learners interact to construct understanding.  Figuring out solutions to tasks with others in your training group will result in a deeper understanding than if the instructor simply demonstrates or gives all the answers.
Something you might have missed, your peer might remember, and vice versa. Help your co-learners out if they are struggling; explaining concepts in your own words helps you consolidate your learning. On the other hand, don’t be afraid to ask others if you’re not sure.
After we have introduced a few features in the acquisition software, we give less detailed instructions (for example – display the data in P1 and visualize the whole negative population) and take a step back. The students help each other remember how to change the displayed data and set a biexponential axis (an axis with negative decades, i.e. below zero).
We also find that students perform better in their second training session if they work with a partner. So buddy up if you’re working on a similar project as another new user.
The key word here is collaboration. It’s not a competition, and you should never compare yourself to others. Everyone is from different backgrounds with different levels of experience and different skill sets. Sharing skills makes us perform better.
6. Be Timely
Don’t book your flow cytometry training session if you won’t touch a flow cytometer again for another six months. You won’t remember anything! Try to organize training within a couple of weeks of your first experiment so that your knowledge is fresh.
We expect that the first and second training sessions will occur within a couple of weeks, and experiments should begin within the same month.
Of course, plans change. So if you aren’t ready to begin experiments, or it has been some time since the last time you touched a flow cytometer, ask your facility if you can have a refresher. At the very least, read through your notes, refresh yourself with the theory, and bring a practice sample to get reacquainted. You don’t want to launch straight into an important experiment and come away with unusable data.
Don’t forget, facility staff are there to help you. Ask if you can have an assisted session but be sure to do this in advance—not 5 mins before your booking as they are unlikely to be available.
7. Reflect Don’t Refract
Reflect on what you have learned and how it applies to your own experiment. The cliché goes, you don’t know what you don’t know! Here are some key elements you should consider:
- What fluorescent dyes will you use, and are they compatible with the cytometers available? Will you need to change any filters?
- What will your gating strategy be? How will you use the combination of antigens to identify your population of interest?
- Are you performing any applications such as cell cycle or calcium flux that may require a specific instrument setup?
- What controls will you need to analyze your data? Any multi-color experiment will require single-color, and FMO controls as a minimum, but there may be controls specific to your experimental setup.
Data acquisition is only one step of a flow cytometry experiment. Are you going to need training and support on other elements, such as:
- Sample prep and staining. The adage in flow cytometry is, ‘Garbage in, garbage out’. You need a good sample prep to get good data.
- Panel design. This means pairing the antigens with fluorophores to achieve the best resolution between populations
- Data analysis:
- In software such as FlowJo or FCS Express.
- High parameter data analysis—creating pipelines with dimensionality reduction/clustering algorithms.
- Data fitting models such as cell cycle or cell proliferation.
- Compensation—identifying and preventing compensation errors.
Make sure you’re on the facility distribution list, so you’re in the loop of any workshops. Sign up for mailing lists such as the Purdue cytometry list, and join societies such as the Royal Microscopy Society (it’s for flow too!) and your regional club.
Tips for Flow Cytometry Training Summarized
You’ll get the best experience in your flow cytometry training if you have a clear idea of your proposed experiment, gain a sound grasp of the theoretical elements, become aware of your gaps in knowledge, and engage in two-way communication with your facility. Good luck and have fun!