Frequently Asked Questions
Basics of Flow
General Facility FAQ
What auxiliary equipment is available in the CAT Facility?
Auxiliary equipment is available in the facility free of charge:
Orbital plate shaker
Orbital plate shaker in a 4°C cold room
Hand held magnetic bead plate washer
BioTek ELx405 automated magnetic bead plate washer
Vacuum manifold plate washer
Vortemp 56 shaking incubator
GeminiBio Moxi V cell counter (fluidic microchip available at cost)
Millipore Scepter cell counter
Smart Tube Base Station
What is the cancellation policy?
We expect the users to let us know as soon as possible when an experiment needs to be cancelled. Cancellation requests must be presented before the start of the reservation. Request for past reservation will not be processed. Cancelling a bench top analyzer reservation: The iLab system allows for online cancellation up to 4 hours before the start of the reservation. Unused booked time will be charged at 80% of the value of the reservation. Cancelling a sorting or mass cytometry experiment: Reservations on the Aria cell sorters can be cancelled by the user up until the point the iLab reservation has been marked as confirmed by the CAT Facility staff. Confirmed sorts and mass cytometry reservations must be cancelled 24 hours before the start of the experiment. Contact the CAT Facility staff to do so. Unused sorting or mass cytometry reservations will be charged in full.
What are your policies for data storage and equipment damage?
I’m writing a manuscript, where to I find information about the instrument I used and acknowledging the facility?
Does the CAT Facility accept samples from institutions outside of UChicago?
Northwestern and the University of Illinois in Chicago are members of the Chicago Biomedical Consortium and as such are entitled to the internal rate.
Basic Flow Cytometry FAQ
Why are there so many different names for "flow cytometry"?
Flow cytometry, FACS, sorters, analyzers, cytometers, mass cytometers, spectral cytometers – what’s the difference??
Flow cytometry and cytometers are general terms that apply to everything listed. FACS does stand for “Fluorescence-Activated Cell Sorting”, however most people use this as a general term that is interchangeable with the term flow cytometry. Sorters and analyzers are specific subsets – the main difference being that cells that have passed through an analyzer end up in the waste container but cells that have passed through a sorter could be collected and used for other experiments. Spectral cytometers are a type of cytometer that collects data on the entire emission spectral of fluorophores – these are available as either analyzers or sorters. Mass cytometers are another type of cytometer that utliizes heavy-metal tagged antibodies instead of fluorophore-tagged antibodies.
How do I know if an instrument can detect my fluorophore?
How do I decide which instrument to use for my experiment?
A basic overview of all services at the CAT Facility can be found on our New Users page.
If you prefer, you can contact the staff to discuss your project and get further assistance in choosing an instrument.
How do I prepare my samples?
There are a lot of factors to consider when preparing cells for flow cytometry:
- obtaining viable, single cells in suspension
- designing a panel of markers and experiment setup and controls
- staining cells with fluorescent antibodies/dyes
- resuspending cells in a volume appropriate for a cytometer
There are a lot of resources available on these topics! First there is an online course called Flow Basics 2.0 that covers the entire workflow of a typical flow cytometry experiment. The Resources Library located under the Resources menu is also a great place to find information. As you’re preparing for your first experiment and starting to get data, be sure to check out the “Avoid Bad Data” series on our blog for some great tips.
Information about resuspending cells is specific to the instrument you’re using – that information can be found on the pages dedicated to each instrument.
What is FACS Buffer?
“FACS Buffer” is a broad term describing the buffer that cells are in as they are stained and run on a flow cytometer. The formulations vary, but anything that keeps your cells happy can be used. The most common basic recipe is:
- 1X Ca/Mg2+ free 1X PBS or HBSS
- Either FBS (0.5-5%) or BSA (0.1-1%)
- Filtered FBS is recommended to remove debris
Other additives include:
- Optional: EDTA (0.5-5mM) if you have very sticky cells
- Commonly used: 1% FBS in 1X PBS (we strongly recommend that you filter FBS or the whole buffer to remove debris form the FBS)
Bicarbonate-based buffers like RPMI could be used, though this may be suboptimal for a small subset of experiments (the sample will come into contact with phosphate-based sheath buffer in the instrument). Regardless of the buffer used, it is critical that it is does not contain a colored additive like phenol red (clear buffers only).
What's the typical workflow of a flow cytometry project?
- Select a cytometer
- Design a panel
- Ensure the tissue of interest can be prepared to contain viable, single cells in suspension (this may need to be optimized)
- Run a practice experiment to determine if data can be acquired as expected (optimize if needed)
- Run experiment and analyze data
- Run more experiments and analyze more data
- Publish data, acknowledge CAT Facility
Many flow cytometry experiments require optimization in order to produce high quality results. The time and effort required for optimization is unpredictable and varies – some projects may be fine without optimization or a single practice experiment whereas complex experiments with large panels may take a month or more to optimize.
Benchtop Analyzer FAQ
Which 5mL FACS tubes should I use?
For the Fortessas/LSR II you must use these 5 mL flow tubes: Falcon #352008. If you do not use these tubes the samples will not pressurize and you will not be able to properly record data.
I'm running samples after hours and there's an issue with the instrument, what do I do?
What are the recommended starting voltages for the fortessas?
For a detailed explanation on how starting voltages were determined, please see this blog post. The recommended voltages should be set up in the default experiment templates on the fortessas. It can also be found here:
Fortessa 4-15 HTS
Attune NxT 4-14
How many cells should I stain? How many cells should I record?
- You need to know how many cells are in your rarest population of interest. Of all the cell subsets you are analyzing which makes up the smallest percentage of the total cells? And what is that percentage – 0.1%? 5%? 40%? The more rare your population is the more total cells need to be recorded. For very rare populations (less than 0.5% total cells), aim to get a minimum of 500-1000 events in that population.
- For general immunophenotyping of immune cell in tissues, 100,000-300,000 is a typical value. For cell lines, 10,000-50,000 is a typical value. These recommendations are general and my not be ideal for your specific experiment.
- Do not assume that you can stain 100,000 cells and then record 100,000 cells. You will not be able to record the entire sample and some cells will be lost during wash steps. Please stain more cells than you need to record.
I can't find my data, what should I do?
- The data was accidentally saved in a subfolder within the folder connected to the data server
- The connection to the server was lost
- You were overly excited about analyzing your data and the server hasn’t had a chance to upload it yet (data pushes to the server about every 30 minutes)
New to the cell sorting service? If you’re planning on using the droplet-based sorters, be sure to read through the information below before your first appointment. Feel free to contact the staff if you have questions!
What should I bring to my appointment?
- Your cells can be brought in whatever media will keep them in good condition – see “What is FACS Buffer” in the Basics of Flow FAQ tab.
- Depending on the nozzle tip used, the cell concentration will vary
- 70um tip (lymphocytes, splenocytes, small cells): 20-25 million cells per mL
- 100um tip (most cell culture, lung and liver cells, etc): 10-15million cells per mL
- At these concentrations, we can sort
- 70um tip: 3mL/hr
- 100um tip: 2mL/hr
- These concentrations will offer an optimal sorting efficiency without impacting sort purity
- The minimum recommended volume is 500uL.
- Use 5mL or 15mL tubes only. Other tubes will not fit the sample chamber.
- For multi-parameter experiments, appropriate single cell controls are highly recommended
- Gating controls, such as unstained cells or FMOs are needed to gate the desired population properly.
- Use 5mL or 15mL tubes only. Other tubes will not fit the sample chamber.
- Collection tubes
- The collection tube will vary with the expected results of your experiment and the downstream application. If you are unsure of which collection tubes to select, please contact CATHelp@bsd.uchicago.edu prior to your sort.
- A wide collection of receptacles can be used, including:
- 15mL tubes (sort 2 populations simultaneously)
- 5mL tubes (sort 4 populations simultaneously)
- Eppendorf tubes (sort 4 populations simultaneously)
- Plates (6-well plates to 384 well plates) -> Available on the AriaII and Aria Fusion only
- Other – contact the CAT facility staff if you have a non-traditional idea in mind or if you want to sort more than 4 populations from your sample.
- The tube should contain collection media to preserve cell viability.
- 1X Ca/Mg2+ free PBS with either FBS (0.5-5%) or BSA (0.1-1%), HEPES buffered (10-25mM of HEPES, pH range 6.8 – 8.2) is recommended.
- Variations might include:
- The addition of EDTA or DNAse to prevent cell clumping
- Higher protein concentration to help viability – you can sort into 50-100% FBS because the sample will dilute the collection buffer (see below)
- Culture media
- Trizol for DNA or RNA extraction
- The sorter will generate an extra amount of volume:
- 70um tip: ~1mL/million cell sorted
- 100um tip: ~3mL per million cells sorted
- If you plan on sorting directly in lysis buffer, be mindful of the dilution impact of this extra volume
- The buffer should be kept at a maximum of 10% of total lysis buffer, or 20-25% if using Trizol LS
- An alternative is to spin your cells after the sort and resuspend directly in lysis buffer.
What should I do at the time of my appointment?
NOTE: For COVID-specifc procedures, see our RRP.
- The CAT Facility operator will be waiting for you at the instrument.
- Your controls will be acquired and gates will be set based on your directions
- A purity check will be run on your samples unless you direct otherwise
- You will need to stay until the gates are set and the purity check is complete. Once the sort is under way, you will be able to leave the facility.
- The Operator will email you at the end of the sort. This may occur when there is no sample left or if the is no more time available. In this case, the operator will present available solutions to complete the experiment.
Tips for Culturing Sorted Cells
NOTE: These tips are for the Arias. For true sterile sorting, you may want to consider the Tyto. Please contact the staff in advance to discuss your Tyto sort with the staff – you may need to alter your panel for the Tyto.
- The Arias are kept as clean as we can, but should not be considered sterile instruments.
- They use sterile 10X ClearSort Sheath mixed with MilliQ water
- The sample probe is bleached between every experiment
- The instruments are flushed with ETOH at the end of every day
- The sheath lines are bleached on a weekly basis or as needed
- Contamination spot tests are taken on all of the instruments on a weekly basis so the staff should be able to inform you of any issue
- The in-line sheath filter is replaced every 6 months or as needed
- The use of antibiotics is strongly recommended. Gentamycin has been shown to be more efficient than Pen/Strep
- We recommend contacting the staff whenever the sorted cells are to be put back in culture. The staff will inform you of any specific issue regarding potential contaminations and will take every step needed to keep the instrument clean.
- Should a sort get contaminated, inform the CAT Facility as soon as possible. It will allow the staff to take the appropriate action needed in a timely matter
Tips for downstream applications involving DNA/RNA
If you would like to lyse your cells, you can either sort them into buffer/media and then pellet them in your lab before lysing them or lyse the cells immediately as they are sorted.
- If you are pelleting cells and then resuspending, keep in mind the centrifuge step will cause some minimal cell loss
- If you are sorting directly into lysing buffer you’ll need to be aware of the volume of the sorted cells:
- For sorting directly into regular trizol (ThermoFisher #15596026), cell buffer amount should be kept to 10% or less. So if you bring collection tubes containing 900 uL of trizol, you can add 100 uL of sorted cells.
- The better option is trizol LS (ThermoFisher #10296010), which is a more concentrated version of trizol intended for liquid samples. The amount of sample added to trizol LS should be somewhere between 25-30% of the total volume. So if you bring 700 uL of trizol LS, you can add 300 uL of sorted cells.
- If using Qiagen’s RNeasy, the ratio is 100uL of sorted cells into 350uL RLT buffer.
- Please discuss with the staff if samples need to be mixed periodically over the course of the sort to ensure all cells are in the lysis buffer
What if there are problems with my sort?
- If you encounter any problems with your sort, please make sure you communicate with the staff. We can’t help you if you don’t let us know. Again, the email is CATHelp@bsd.uchicago.edu
- Cancellation policy: The sorters are billed on the amount of time that was booked or used, whichever is greater. Sorting reservation can be cancelled by the user on iLab up until the point where it has been approved by the core. At that point, you will need to contact the staff to cancel your time. Unused sort time will be billed in full.