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Why Flow Cytometry Fluorescence Compensation Is Critical For Quality Measurements

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Written by Tim Bushnell, Ph.D

Fluorescence compensation is one of the more difficult, understandably confusing, and misunderstood aspects of flow cytometry.

Compensation is almost always necessary when more than four or five colors are measured simultaneously. As such, understanding what compensation is, why it is necessary, and what to expect when using it, are critical for generating useful and high-quality data from flow cytometry experiments.

In order to understand compensation correctly, you need to understand the answers to 3 core questions related to compensation…

Fluorophore multicolor cytometry compensation

1. What is the nature of fluorophores?

The necessity of compensation arises from the nature of fluorophores, the fundamental and cherished tools of flow cytometry.

We often tend to associate a fluorophore with a particular color. For example, we often say that “FITC is ‘green’” and “APC is ‘red.’” While it is true that when FITC is excited it is most likely to emit photons in the “green” range (around 520 nm), it is also true that FITC will emit photons of other wavelengths of colors, fro ...

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How To Get A Flow Cytometry Job In 5 Steps

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Written by Tim Bushnell, Ph.D

Flow cytometry is a powerful technique impacting both clinical and research.

If you’re looking for a career, flow cytometry technology can take you many places. An experienced flow cytometrist can find a job in a biotechnology company, in academia, or in a clinical setting.

Rather than focusing on specific career information, I would like to highlight one career option that has been very good for me since my transition from research scientist to core manager over a decade ago.

Core facilities, or Shared Resource Laboratories (SRL) as this Cytometry A paper made popular, represent an investment by institutions in resources and personnel.

Staff and directors working in SRLs have the advanced training and experience to support the researchers and research mission of the institution.

Working in an SRL is a different and very exciting career option for researchers who enjoy flow cytometry, enjoy working on many different projects, and enjoy working in scientific customer service.

That being said, starting out in an SRL can be daunting. Professionals of al ...

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Flow Cytometry Procedure For Accurate Sorting Of 5-10 Micron Cells

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Written by Tim Bushnell, Ph.D.

In the most common applications, cell sorters are utilized to purify cells on the order of about 5-30 microns in diameter, often human or murine leukocytes, or cell lines derived from liquid or solid tissue types.

Considering this typical size range, cells between 5-10 microns in diameter are typically the simplest cells to sort. That being said, the general rules of thumb in forming sample preparation, instrument setup, and sample collection are critical to generate sorts of high purity, recovery, and viability — the hallmarks of sort quality.

To this end, there are 4 steps you must take to ensure high quality when sorting 5-10 micron cells by flow cytometry…

Minimize blood sample debris for accurate flow cytometer measurements

1. Protect cell sample quality.

The tried-and-true adage of flow cytometry — garbage in, garbage out — should hover above and firmly guide each step of the sample preparation process for any kind of cytometry experiment, but is especially critical for sorting.

Poor sample quality will most surely guarantee sacrifices in purity, recovery, and/or viability, often to the extent of signi ...

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How To Annotate Your Data With FlowJo Keywords

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Written by Tim Bushnell, Ph.D.

Do you annotate your data properly?

If you don’t, you’re not alone. Most people don’t take advantage of the keywords embedded in the FCS (Flow Cytometry Standard) files to annotate their data. FlowJo makes this easy to do.

First off, what are keywords and why do you want to use keywords?

The flow cytometry standard file formats (FCS) was developed by the International Society for the Advancement of Cytometry (ISAC) to create a standard file format for all cytometers to use. The purpose of this was so that no matter who manufactured the instrument, or the analysis software, files could be created and read in any system.

As part of this FCS standard, ISAC created a set of core keywords that MUST be used. These include information on the parameters, scaling, FCS file format, date, file name, and numerous others. You can read more about the FCS file format here.

When it comes to annotating your flow cytometry data with keywords, there are 3 things you need to know…

1. Certain keywords are “required” by the Flow Cytometry Standard (FCS). ...

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Flow Cytometry Protocols To Prevent Sample Clumping

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Written By Tim Bushnell, Ph.D.

Good flow cytometry depends on a high-quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze.

Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Thus, sample preparation becomes the critical first step in any flow cytometry experiment.

As Henry Ford once said, “Before everything else, getting ready is the secret of success.”

For the purposes of this article, our attention will be focused on mammalian cells and cell-lines. While other samples can benefit from these tips, samples from the environment, bacterial and yeast samples, and small particles like extracellular vesicles, have their own issues and tricks to consider.

Lysis solution flow cytometry protocol

To get high quality results, follow these 3 sample preparation steps…

1. Get the purest sample possible.

Taking the example of working with peripheral blood, one of the first and most important choices to be made when using peripheral blood is the type of ...

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