3 Experiments You Can Do Easier On An ImageStream Flow Cytometer

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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

Antibody Panel DesignSudoku puzzles seem to be all the rage.

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

Manual Compensation Spillover(Figure courtesy of Pratip K. Chattopadhyay, Ph.D.)

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

5 Flow Cytometry Errors Reviewers Despise

We all know that flow cytometry makes individual measurements on large populations of cells, it allows for statistical analysis of the data, lending strength to a researcher’s conclusions.

Likewise, the isolation of very complex populations by flow cytometry cell sorting can help lead to a richer understanding of the intricate biology at the genomic, proteomic and functional level.

As a reviewer of papers and grants, I am always especially interested in the details of HOW the experiments were performed because that is the critical foundation for what the data is able to tell us–and what it can NOT tell us.

Mistakes That Mark You As Untrained

It may sound harsh but there are some errors that reviewers hate seeing. The good news is that by learning what reviewers dislike (or even despise), you can learn how to run better and more accurate experiments. Here are 5 errors... Read More

The Most Common Mistake Researchers Make When Designing Flow Cytometry Antibody Panels

Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help.

With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors.

Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation... Read More

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