Cytometry and Antibody Technology

Benchtop Analyzers

Full Spectrum and Traditional Flow Cytometers

Check Instrument StatusAnalyzer FAQ

Full Spectrum Flow Cytometers

Full spectrum flow cytometers (FSFC) allow the measurement of fluorescence generated by dyes or fluorophores excited by different laser beams. Unlike traditional flow cytometry where each parameter is allocated to a specific detector, the FSFC utilize a greater number of detectors then the number of measured parameters and no specific filter is dedicated to any given signal. Instead, the instrument measures the entire emission of each signal generated and unmix the signatures during the sample analysis. This allows for the study of a larger number of parameters simultaneously.

Aurora

Model: Aurora
Manufacturer: Cytek Biosciences

2 identical units available – called Blue and R (a colored piece of tape is used to differentiate them.)

Spatially separated lasers
– 355nm 20mW
– 405nm 100mW
– 488nm 50mW
– 561nm 50mW
– 640nm 80mW

Plate Loader
– Aurora-Blue: Auto Sampler – acquire samples from96-well or 384 well format.
– Aurora-Red: Automated sample loader (ASL) – orbital shaker and supports 40 tube racks, 96 well standard height plates, and 96 well deep plates.

Absolute cell count

For more information about the Aurora, please see our resources page

Traditional Flow Cytometers

Flow cytometers measure the emission of fluorophores and dyes excited by various laser beams. Each signal is directed through an array of mirrors and filters to a specific detector. The number of lasers and the filter configuration can vary between instruments. You will find a full description of each instrument below. More information on recommended fluorophores, brightness indexes, and recommended voltages can be found on the instrument guides page and the panel design page.

The Fortessa X20 will not be repaired. It will be replace by and Agilent Penteon (5 lasers/30 parameters)

 

Model : LSR Fortessa
Manufacturer: BD Biosciences

Spatially separated lasers.
– 488nm 50mW
– 405nm 50mW
– 561nm 50mW
– 640nm 40mW

Optical configuration: Fortessa 4-15

Located in BSLC room R022

Model : LSR Fortessa
Manufacturer: BD Biosciences

Spatially separated lasers.

– 488nm 50mW
– 405nm 50mW
– 561nm 50mW
– 640nm 40mW

Optical configuration: Fortessa 4-15 HTS

Plate loader

Located in BSLC room R022

Model: Attune NxT
Manufacturer: ThermoFisher

Spatially separated lasers
– 488nm 100mW
– 405nm 100mW
– 561nm 140mW
– 640nm 140mW

Optical Configuration: AttuneNxT 4-14.PNG

Plater loader

Absolute Cell count

Acoustic Focusing

ThermoFisher Training material

More information

Located in BSLC room R022

Model: BD LSRII
Manufacturer: BD Bioscience

4 spatially separated lasers

– 488nm
– 405nm
– 561nm
– 630nm

Optical Configuration: LSRII 4-12

The manufacturer may no longer support this instrument beyond 2022

Located in BSLC room R022

 

Benchtop Analyzer FAQ

Basic Questions

How are the analyzers named?

The nomenclature reflects the instruments as listed in the CAT Facility iLab’s site: Model_# lasers-# detectors. For the full spectrum flow cytometers the CAT Facility assigned colors to distinguish them because they are otherwise identical.

How do I decide which analyzer to use?
  • If you are just looking for an overview of which fluorophores can be used on each cytometer, see the Analyzer Comparison Table.
  • If you need further assistance, please contact CATHelp@bsd.uchicago.edu and the staff will help you decide which analyzer is best for your experiment.
What is the configuration of the benchtop analyzers?

This information can be found here and the button above the Analyzer FAQs section:

This table contains the laser lines and bandpass filters for all of our analyzers. It also contains a list of popular fluorophores that can be assigned to each detector. Only one fluorophore per row should be used in a panel of antibodies, if you choose two or more fluorophores from a row you will not be able to distinguish them.

I've never done flow cytometry before. Where to I get started?

The CAT Facility staff is here to help you! Here’s a general overview of steps:

  1. Complete CAT Facility training. You can access more information about that on the New User page.
  2. After completing the required Flow Basics Course, you may find the Flow Basics 2.0 course helpful (optional, located in Additional Courses under the Training menu).
  3. Pick the analyzer appropriate for your experiment and be sure to be trained (for those wanting unassisted use). The staff can help you if you’re unsure!
  4. Understand how to prepare your sample (acquire single cells in suspension) and get a protocol to stain your cells. Resources can be found through our menu bar – see our sample preparation page.
  5. Plan your panel. Resources can be found through our menu bar – see our panel design page.
  6. See the next section of this FAQ for information regarding setting up the instruments.

Please do not hesitate to contact the staff if you have any questions. We also offer consultation services for more extensive assistance.

Using the Analyzers

How many cells should I stain? How many cells should I record?

These questions are entirely dependent on your experiment. For an in-depth explanation, please see this blog post.

Simple answer:

  • 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.
How should I resuspend my cells to run on the cytometer?
  • If you are new to flow cytometry, it is safer to resuspend your cells (especially controls) in a larger volume. This will allow you to properly set the instrument settings without running out of sample
  • As you become more comfortable with the cytometer, you can use more concentrated controls so that they run faster on the instrument
  • The volume to resuspend cells in depends on the instrument
  • Fortessas
    • 150 µL absolute minimum, 300-500 µL minimum for new users, 150-300 µL for advanced users
    • New users need extra time to set up controls – consider running your controls more dilute so you have time to adjust settings
    • It’s best to bring concentrated samples, once you are at the cytometer you can easily dilute your tubes if they are running to quickly
    • In general, run samples at less than 35,000 events/sec
  • Attune:
What tubes do I need to bring my sample in?

The Fortessas require a specific 5 mL FACS tube: Falcon #352008. If you use a different tube or the tube is cracked, the tube will not pressurize and you will not be able to run your sample. The Fortessa 4-15 HTS has a plate loader that will accept 96-well plates.

The attune can accept 1.7 mL eppendorf tubes, 5 mL FACS tubes, or any similarly sized tube that fits into the sample loader. It also has a plate loader that accepts 96-well plates.

The auroras accept 5 mL FACS tubes and have a 96-well plate loader. We find that using the tube inserts for small volumes are not really required and require an extra setup step.

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

Fortessa 4-15 HTS

Attune NxT 4-14

Pricing