Flow Basics 2.0
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Flow Basics 2.0 is a series of courses that builds on the original Flow Basics course. This series outlines all of the practical steps for setting up a flow cytometry experiment and highlights factors that impact final results. The videos can be watched on our YouTube channel and the slides can be downloaded below.
There is also a good series of blog posts called “Avoid Bad Data” and a list of Best Flow Cytometry Publications in 2022.
Flow Basics 2.1: Staining Protocol and Reproducibility
- Overview of a basic cell surface marker staining protocol
- How does Fc block work
- Five factors that affect antibody staining
- What is the difference between the available viability dyes
- Fixation
- Download Flow Basics 2.1
Flow Basics 2.2: Optimizing the Staining Protocol
- Why you should optimize tissue digestion
- Alternative blocking options
- How to determine the number of cells to stain for an experiment
- Why and how to perform antibody titration
- Download Flow Basics 2.2
Flow Basics 2.3: Panel Design
- Information needed before beginning panel design
- Tools available for for panel design
- How to assign markers to fluorophores
- Which fluorophores to avoid
- Strategies for placing high spillover fluorophores
- Download Flow basics 2.3
Flow Basics 2.4: Experiment Design
- What controls to consider and when to use them
- Single Stain
- FMO
- Isotype
- Isoclonic
- How to divide cells between controls
- Bonus video: How to pipette FMO controls
- Download Flow Basics 2.4
Flow Basics 2.5: Instrument setup and Automated Compensation
- Why organizing the experimental layout is useful
- Shortcuts for labeling tubes quickly in BD FACSDiva
- How to set voltages on the cytometer
- How to properly use automated compensation tools and avoid compensation errors
- How to identify compensation errors
- Download Flow Basics 2.5
How to Identify Bad Flow Cytometry Data – Bad Data Part 1
When the global pandemic hit and Chicago’s shelter in place orders prevented me from going into the lab, I started doing some experiments in my kitchen instead. I decided to take on baking sourdough bread (like many other quarantined individuals). And while I...
Laura Johnston | Sep 30, 2020 | 26 Comments
How to Identify Problems with Flow Cytometry Panel Design – Bad Data Part 2
In the first post of the "bad data" series, I mostly discussed how to identify problems with cytometer settings and compensation by looking at the data plots. (Read that post here in case you missed it!) In this post I’d like to expand on that by going over common...
Laura Johnston | Nov 19, 2020 | 3 Comments
How to Identify Problems with Flow Cytometry Experiment Design – Bad Data Part 3
Two weeks ago I covered my red flags for identifying bad panel design (read that here if you missed it!). This week I’ll go over my red flags for experiment design, which mostly means controls. Controls are needed to be able to appropriately interpret experiment...
Laura Johnston | Dec 3, 2020 | 8 Comments
How to Fix Flow Cytometry Compensation Errors (and Unmixing Errors) – Bad Data Part 4
I have an unpopular opinion: I love compensation. Usually when I bring up compensation I’m met with a chorus of frustrated noises. But to me compensation is an opportunity to solve a complicated puzzle – and I really love a good puzzle. In this post I’m going to...
Laura Johnston | Aug 9, 2021 | 0 Comments
Cleaning up your flow data with algorithms – Bad data part 5
It's time to get back to the Bad Data series. I'll use a data set I had to troubleshoot for one of my users to illustrate the importance of cleaning your data. We mentioned FlowAI and FlowClean in the first installment of this series, and this post will hopefully...
David Leclerc | May 10, 2023 | 2 Comments
