Flow Cytometry Education | Department of Immunology

Upcoming Seminar(s)

Currently working with FluoroFinder, Flowjo, and Cytek for the next few seminars.
Please send requests for future seminar topics to flowcore@pitt.edu.
Recordings and/or slides from past seminars can be found at the bottom of this page under "Unified Flow Core Seminars."

Background on Conventional Flow:
ThermoFisher: https://www.youtube.com/watch?v=sfWWxFBltpQ
UChicago https://voices.uchicago.edu/ucflow/training-courses/ (download Flow Basics PPT from here) Although at first it seems to go over many of the same topics as the ThermoFisher video, it then goes deeper... introducing spectral flow cytometry, data analysis, and ways to clean up your experiments/data.
Other Flow resources at ThermoFisher.
Other Flow resources at UChicago
BD Courses (many are free/online)
Introduction to Flow Cytometry - Aarika MacIntyre - 2021
Intro to Advanced Flow Cytometry - Florian Weisel - Fall 2021
LSR II and Fortessa Experiment Guide
How to run experiments in Diva. (handout)
LSR/Fortessa Course at UChicago
Scroll down for video on how to use Diva, how to fill sheath, and troubleshooting guides.
By University of Chicago's Cytology and Antibody Technology Facility.
Troubleshooting and Advanced Declogging (LSR II & Fortessa)
Requires Pitt Login. (turn down volume)
Also see Troubleshooting Guide at UChicago (skip down to bottom 25% of page or ctrl-f (command F on Mac) and search for "Troubleshooting."
FluoroFinder
Get a free account as a member of Pitt. Very helpful for designing panels for specific machines. Can design spectral panels here as well, but Cytek Cloud has nicer tools specific to spectral experiments.

Cytek (Aurora) Biosciences Resource Page
Cytek Cloud - Good place for spectral panel design for Auroras (all of ours are 5 laser). Can even save an experiment template to save time setting up at the machine! Account is free.
Aurora Training Course
This is an EXCELLENT course on spectral flow cytometry by the University of Chicago's Cytology and Antibody Technology facility.
Unified Flow Core
Aurora Reference Guide
Be sure to check out the AF Explorer Tool seminar from November 2023.

We are currently reconfiguring the Zeiss microscope to work with Micro-Manager and will soon offer training to run experiments using this software. Micro-Manager is also ImageJ based and works well with QuPath.
Micro-Manager 2.0 user's guide.
Canopy ZellScanner Tutorials and Guides
Canopy ZellScanner Publications/Webinar Page
Webinars require a free account to access.
Antibody Resources/Guidelines (Easiest Way):

Do a PubMed search for papers using the following terms: multiplex imaging, chipcytometry (one word), etc...
Look for papers that use the ZellScanner, your tissue type (frozen or paraffin embedded), species, and tissue of interest (if possible).
Check out the methods section for a table of antibodies used, with concentrations, which you will use as a STARTING POINT for your project.
Note that the Canopy ZellScanner Publications/Webinar Page also lets you search publications.
If you can't find an example of someone using your antibody on the ZellScanner, don't despair, email the Flow Core and we can assist you.

Data Analysis Resources (Chip Cytometry)
You're going to want to analyze your data in QuPath (free), which is based on ImageJ, but is designed for large images, including those from multiplex experiments.
Download QuPath from https://qupath.github.io/ For best results, install it on a workstation, as opposed to an underpowered desktop or laptop. We have a copy on the analysis computer if needed.
Note the VALUABLE resource links under the download link on the QuPath page, including the forum, docs, YouTube channel, etc... An excellent video to start with is this one: https://youtu.be/WTAgXpuuqNY Be sure to look at the valuable links in that video description as well.

Flowjo
Note: no account needed to view educational/reference materials. Flowjo can be used to analyze spectral and conventional flow data.
Flowjo Courses
Webinars Scroll down to "Recorded Webinars" if you aren't able to attend a live one.
Flowjo Exchange Download various (free) plugins here. Especially recommended are: FlowAI, FlowSOM, ClusterExplorer, and check out ezDAFI. Recommended dimensional reduction plugins in addition to TSNE (which is now part of Flowjo), UMAP, FitSNE (faster TSNE)
The support staff at Flowjo is very responsive and helpful. Feel free email them with questions about the software, plugin installation, or the plugins themselves: flowjo@bd.com
Check out the next section as well for background on dimensional reduction. Most tools mentioned are now available as Flowjo plugins.
ImageStream Data Analysis
IDEAS - analysis software manual
IDEAS Tutorial Series is available on Analysis Computer in Assembly G532
Machine Learning Manual - IDEAS v6.3 has the machine learning module (this version is available on analysis computer in Assembly G532).

Sorting Seminar - Nicole Brandon, M.S. - January 2024
Slides
Recording will be posted after transcript edits are complete (still working on it).
New Autofluorescence Explorer tool for spectral data on collected Cytek Auroras - Dr. Paul Dascani - November 2023
Presentation - Note the chapter divide to skip to software demo section. And I edited the transcript for you, so you can turn that on if needed.
Raw NxN Worksheet for Spectroflo (This worksheet is available on the Desktops of the various Auroras.) Download and open in Spectroflo to use it.
Handy list of common fluorochromes and their peak fluorescence channels on 5-laser Cytek Auroras (as all of ours are).
ImageStream (webinars might be limited to Pitt users only)
May 2023
July 2023
Chip Cytometry (ZellScanner)
Intro to Chip Cytometry - Dr. Lisa Borghesi - Spring 2022
Flow Cytometry... With Tissue??? - Nicole Brandon - Spring 2022
Intro to Advanced Flow Cytometry - Dr. Florian Weisel - Fall 2021
Conventional vs. Spectral Flow - Dr. Lisa Borghesi - Fall 2021
Aurora Panels (requires Pitt login)
Introduction to Flow Cytometry - Aarika MacIntyre - 2021