How To Perform A SPICE Analysis With FlowJo
Gone is the rule of 2-3 color experiments. Even beginners are starting with 5+ color assays, and the adoption of mass cytometry has the potential to increase our headaches even more.
Current data analysis methods are good for single tubes or small cohort studies. What do you do when you have a large dataset, with multiple sampling conditions, and multiple outcome measurements?
With data complexity of this nature, one can export the numerical data to a third party analysis package, but even then the analysis can be difficult to perform.
To overcome this limitation, and to allow for better discovery science, Mario Roederer and his colleagues have developed a solution. SPICE was developed... Read More
Why Recovery, NOT Purity, Is The Best Measure Of A Cell Sorting Performance
(Written by Carol Oxford, ExCyte Founding Partner.)
One of the best perks of being a part of the flow cytometry community is the opportunity to attend one of the many regional flow meetings.
The chance to network with colleagues that do what you do is incredibly energizing.
It’s also a great opportunity to see what new research ideas they’re involved in before they’re published, and to talk with them directly about their ideas, and the research they’re involved in. I was recently at the Australasian Cytometry meeting and had a chance to talk to Dr. Rui Gardner, the Director of Flow Cytometry at the Gulbenkian Institute of Science in Lisbon, Portugal.
Rui gave a great talk on the concept of Rmax, which I’ll be talking about here. I want to make sure and acknowledge the other colleagues also involved in this work, who are Andy Riddell, Flow Cytometry Manager at Wellcome Trust-MRC Stem Cell Institute, Centre for Stem Cell Research University of Cambridge; Alexis... Read More
3 Experiments You Can Do Easier On An ImageStream Flow Cytometer
What happens if one combines the power and speed of traditional flow cytometers with the resolution of a microscope?
Cytometry is the study of biological processes at the whole cell level and includes techniques like light microscopy and electron microscopy.
But microscopy by itself is a bit different.
From the earliest days of microscopy, including the use of the first true microscopes by van Leeuwenhoek and others, scientists have been able to start seeing the finest details of a cell.
With the development of the flow cytometer,... Read More
How To Create The Right Flow Cytometry Antibody Panel Every Time
I see it in coffeehouses, at the airport, even in doctors offices. Everyone is trying to work out how to fit the numbers into the grids so that everything adds up properly.
Designing polychromatic flow cytometry panels is much like the Sudoku puzzle.
In this case, the grid is composed of the antigens on one side, and the cytometer detectors on the other.
The goal is to fill in the grid correctly.
Instead of adding up to 45, like in Sudoku, the flow cytometrist is trying to optimize the ability to make a sensitive measurement to answer the biological question the researcher has set out to answer.
Solving the Polychromatic... Read More
Why You Should Never Manually Compensate Your Data
One of the most important steps in proper flow cytometry is the process of compensation.
There are a lot of rumors and mysteries that fill laboratory notebooks about the process. Some of these processes are correct while others lead to incorrect compensation, resulting in poor data.
Compensation is the process for correcting for the spillover.
Spillover is the overlap of a fluorochrome into a second channel due to the physics of fluorescence. This is a mathematical value that ensures that contributions from the fluorochromes not being displayed are not affecting the distribution of the data being displayed.
One of the most common rumors or practices... Read More