Frequently Asked Questions

1. Attend the Introduction to Flow Cytometry lecture series (offered monthly). Sign up here.
2. Create an account in the iLab scheduling system

The Flow Cytometry Lab has two locations:

• Wisconsin Institutes for Medical Research (WIMR) Room 7016 (laboratory) and 7003 (office)
• UW Biotechnology Center, Room 2360

See our Equipment & Pricing page.

Does the Flow Lab staff offer training on instrumentation? 

Yes. All customers who plan to operate an analysis instrument independently must complete our Introduction to Flow Cytometry series (see more information at https://cancer.wisc.edu/research/resources/flow/facility/#intro) and complete two hands-on training sessions for the instrument platform you intend to run. There are dedicated training times set aside on each instrument each week; see our iLab scheduling page for details. 

I’ve been doing flow for years. Do I really need to attend the introductory class? 

Yes. We continually update the class content and tailor it to our instrumentation. We also encourage customers to re-take the seminar if they’d like a refresher, and we offer a variety of other seminars throughout the year. 

I’m already trained on the Attune. Can I make appointments on additional platforms? 

You will need to do hands-on training sessions for each platform you wish to run. However, you do not need to take the introductory class again. 

Do you offer training on flow cytometry data analysis software? 

We can answer questions during our weekly data analysis office hours. These walk-in sessions take place in our computer lab in CSC K4/532, on Wednesdays from 10am to noon. We can also set up other times to work with you, and refer you to online resources. 

Which instruments share/overlap training sessions? 

Each instrument type runs a different acquisition software and requires separate training. In cases where we have multiple instruments using the same software, you are only required to train on one of those instruments to run any of them. 

For the Attune NxT cytometers (Thunder, Lightning, River), sign up for training on Thunder or Lightning. 

For the Aurora cytometers (Pinky and Clyde), sign up for training on Clyde. 

How many people can attend a single training session? 

We can train up to 3 people from the same lab at one time. For group training, have one person book the appointment and enter the additional name(s) in the Notes section of the reservation form. 

How do I decide which instrument to use? 

If you need certain subpopulations returned to you for downstream applications, then you must choose a cell sorter. If you need a UV laser to excite any of your fluorochromes, then choose the Fortessa or Aurora. We’re happy to talk with you about your experiment to suggest an instrument as you design your panel. 

Do I need to schedule an appointment to use Core services? 

Yes. Every customer using equipment or services in the Flow Lab must make an appointment in advance. We suggest booking appointments at least a week in advance. The longer the appointment you need, the farther in advance you should reserve time.  

We suggest booking 1.5-2 weeks in advance for cell sorting appointments. 

Self-run appointments on the analyzers can be booked at any time, as long as the start time hasn’t passed yet. Reservations for training, staff-assisted appointments, and cell sorting must be made at least 24 hours in advance of the start time. 

We realize that science is sometimes unpredictable. If you want to request a last-minute appointment, please call us. We will do what we can to work you into the schedule. 

How do I schedule an appointment to run samples? 

Use the iLab scheduling tool to make appointments: https://uwmadison.ilabsolutions.com/account/login 

Any customer can book a cell sorting appointment during our sorting hours, provided the appointment is booked at least 24 hours in advance of the start time. Appointment requests will be cued for staff approval. 

Any customer can book a training or staff-assisted appointment on an analysis instrument during that instrument’s designated weekly training time. These days and times are listed in our iLab scheduling page. If your experiment timeline requires staff assistance outside of the training blocks, please email our staff at least one week in advance to request help with your situation. 

If you have completed hands-on training on a particular instrument, you may book that instrument during its hours of availability. 

Can I have all of my experiments run by the same staff member? 

No. Eventually, that person will go on vacation, be sick, or leave the lab. We train all staff members to be able to run samples and produce good results. We can work with you to standardize instrument setup using acquisition templates to reuse gates and beads to standardize detector sensitivity. 

Can I have all of my experiments run on the same instrument? 

Yes. In many cases, there are good reasons to do this. Book your appointments well in advance to be sure you can get time on the instrument on the days you’ll need to run samples. 

How do I schedule a meeting with a Flow Lab staff member? 

We love to talk about science! Reach out to us by email to set up a meeting about your experiments. 

Why can’t I see the whole schedule for the instrument I want to use? 

You must complete hands-on training (with fluorescent beads) and a staff-assisted appointment (with your cells) before being designated as a trained user on the instrument and permitted to book self-run appointments. Each instrument type requires separate training. If you have completed the required training sessions and still can’t access the full schedule, contact us. 

How do I fix my cells? 

There are a variety of good resources for protocols, including supporting information from OMIPs and artcles from Current Protocols in Cytometry.  

First, be sure to create a good single-cell suspension. If the cells are clumpy to start with, fixation will make those clumps impossible to dissociate. 

A typical starting point for formaldehyde fixation is 15 minutes at room temperature in 1-2% formaldehyde. 

How long can I store my fixed cells before analyzing them? 

Several factors contribute to the stability of samples after fixation. In general, the sooner you run them, the better.  

Formaldehyde-fixed samples should be washed into DPBS with some protein, not stored in fixative. Try to analyze the cells the next day, or at least within a couple of days. The first signs of degradation are typically “collapsing” populations on the FSC/SSC plot as the cells shrivel up. Tandem fluorophores may also degrade during prolonged storage. 

If you are planning a long experiment and want to store samples, do a pilot run in which you test various durations of storage to see how long your particular samples will last. 

If you use ethanol to fix the cells (typically for DNA content analysis), wash the samples into PBS and cap them tightly to prevent evaporation. Store them in the dark at 4C. They’ll last for months. 

What’s your favorite viability dye? 

For most experiments with live cells: DAPI 

For most fixed cells or panels where other fluorochromes overlap with DAPI: GhostRed780 

For spectral cytometry experiments: it really depends on the rest of your panel 

Not sure what to choose? Tell us more about your experiment and ask us! 

Do you provide antibodies or other staining reagents? 

Generally, no. We use very few staining reagents ourselves. There is too much variation in species/marker/fluorochrome combinations across our customer base for us to keep stocks for use. In some circumstances, when you’re waiting for a backordered reagent, we might be able to find something suitable or connect you with someone who has it. It doesn’t hurt to ask – just know that it is a long shot.  

We do keep stocks of viability dyes and antibody capture beads which we can aliquot for you if you’re running a pilot experiment or waiting for an order to arrive. 

Do you provide any reagents or consumables? 

We do buy some items in bulk and pass the cost savings on to our customers. These include: 

• Spherotech Rainbow Fluorescent Particles Mid-Range (Cat. RFP-30-5A) for standardizing assay/instrument setup 
• 96 well deep-well plates compatible with the Attune NxT AutoSampler 
• Disposable Counting Chambers for Nexcelom Cellometer Auto T4 Bright Field Cell Counter and the ThermoFisher Countess 3FL Automated Cell Counter 

What should I bring to the Flow Lab for my appointment? 

Bring your samples already stained with all antibodies, dyes, and other reagents needed for your experimement. Filter your samples immediately before coming to the lab, or come a little early and filter them when you get here. Be sure you have all your controls.  

If your appointment is for cell sorting, you must also bring collection tubes/plates containing appropriate collection medium. 

If you have questions about sample tube or media selection, please contact the core staff. 

Do I need to filter my samples? 

Yes. Flow cytometry requires a single cell suspension flowing smoothly through the laser beams. Clumps in the sample will disturb this flow. This can result in inaccurate measurements or clog the instrument. Samples should be filtered after the last centrifugation step, ideally immediately before loading the samples onto the instrument. You can use your own filters, or use filters in our prep room. 

Remember that filtering will not break the aggregates into single cells; it will remove those aggregates from the suspension. You will get more data by creating a good single cell suspension during your sample preparation. Additionally, filtering will not remove doublets from the sample; you will need to apply a singlet gate in your gating strategy to restrict analysis to single cells. 

How do I access my data? 

All data generated in the Flow Lab must be exported from the data acquisition software onto the Flow Lab data server. Each customer must have their own login credentials for the data server. Computers from the Department of Medicine will display the data server as a mapped drive when an appropriate login is used. Other computers can access the data server by using FILR; more information can be found here https://cancer.wisc.edu/research/resources/flow/flow-lab-data-server/.  

Does the core provide Flow Cytometry data analysis software? 

We have a computer lab in CSC K4/532. If the door is locked, contact the Flow Lab staff for access. A variety of data analysis software programs are installed on these computers. 

Customers have two options to purchase software licenses through the Flow Lab. Both options are annual licenses billed once per year through iLab along with your Flow Lab usage: 

FCS Express is an email-based license. You may download the software to multiple computers, and log in on one computer at a time to analyze data.  Each license costs about $234 per year, billed in October. Find more information at https://cancer.wisc.edu/research/resources/flow/fcs-express/. 

FlowJo licenses can be email-based or hardware-based. Each license costs about $220 per year, billed in March. Find more information at https://cancer.wisc.edu/research/resources/flow/flowjo/. 

Do you archive data, and how can I access old data? 

This Flow Lab data server is designed for temporary data storage, and we reserve the right to remove data after 1 year of storage. Each lab must transfer their data to their own storage drives for long-term storage. We clean data off each acquisition computer every Monday. Experiment files are backed up locally for approximately 3 months. Contact Flow Lab staff for help if you need to access these backups. 

Why are there so many passwords? 

Because we care about your digital security! 

Appointment scheduling in iLab requires each person to have a unique login (usually a UW NetID). 

Instrument software logins are based on the lab/PI name, and passwords are shared among lab members. 

The data server is administered by Department of Medicine IT and requires each individual to have a unique login. The username is assigned by DoM IT and may be different than your UW NetID. This password must be changed at least once per year. To change your password, visit https://www.medicine.wisc.edu/password. For help with login credentials and password issues, please contact help@medicine.wisc.edu. 

How can I gain access to the core resources after hours and on weekends? 

Customers who have completed hands-on training and gained staff approval can request access to the Flow Lab for evenings and weekends. Fill out this form to request access to our WIMR and/or BioTech locations. 

Which instrument is the best? 

Each platform in the Flow Lab has its own strengths and limitations. Contact us to talk about your experiment so we can make a suggestion. Factors we consider include the number and nature of fluorochromes in your current experiment, the possibility that you might expand the staining panel in the future, the number of times you’ll do the experiment, and the speed and typical schedule availability of the instruments. 

I want to do something new with a flow cytometer. Will you help me? 

We’re always excited to talk about new projects and ideas. We also use conversations like this to guide our plans for purchasing and upgrading equipment. However, due to the number of researchers depending on our instrumentation, we cannot do significant after-market modification to an instrument that could compromise its performance for other customers.