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How To Differentiate T Cell State With Flow Cytometry

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T Cell state differentation using flow cytometry

Written by Jennifer Snyder-Cappione, Ph.D.

Have you ever wondered what has gotten into your T cells?

Are they activated, resting, tolerant, senescent, anergic, exhausted, or maybe just having an off day?

During short-lived immune responses, such as responses to acute infections or vaccines, there are three classically defined T cell states:

  1. naïve,
  2. activated memory, and
  3. resting memory.

Here’s how to determine whether your T cells are naive, activated or resting memory, or exhausted, and how effector function plays into that.

1. Naïve cells.

Naïve T cells have not encountered an antigen and secrete IL-2 and some chemokines. However, antigen-experienced (memory) T cells, have differentiated into effector cells that can produce cytokines besides IL-2, such as IFN-g, IL-4, and IL-17.

Markers used to identify naïve T cells include CD45RA and CD62L in human and mouse samples, respectively, with CD45RO (human) and CD44 (mouse) present on memory T cell populations.

It is best to use functional profiling as the primary determinant of T cell state in these instances, with surface ...

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How To Use Flow Cytometry To Analyze Rare Cells Within Heterogeneous Samples

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Written by Tim Bushnell, Ph.D

The power of flow cytometry is in the ability to analyze thousands of events rapidly so that finding the proverbial needle in the haystack of heterogenous cells can be done relatively easily.

The discoveries that flow cytometry has enabled because of this ability cannot be underestimated. Prior to flow cytometry, finding rare cells, let alone studying them, was a massive undertaking.

Consider the comparison (and warning) that the great inventor Nikola Tesla once expressed…

“If he had a needle to find in a haystack, he would not stop to reason where it was most likely to be found, but would proceed at once with the feverish diligence of a bee, to examine straw after straw until he found the object of his search… just a little theory and calculation would have saved him ninety percent of his labor.”

Flow cytometry provides one with the theory, calculation, and procedure they need to find a needle (rare cell population) in a haystack (whole blood, cell sample, etc.).

If you’re getting into rare event analysis, there are some additional consi ...

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How To Troubleshoot The Flow Cytometer Fluidics System

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Written by Tim Bushnell, Ph.D

Flow cytometers have three major components:

  • Fluidics that move the cells from the sample tube to the intercept point
  • Optics that collect the light
  • Electronics that convert the photocurrent into a digital value that is stored for later analysis

If we look at the fluidics system of a standard flow cytometer, laminar flow is established by the sheath fluid. This is even flow with fluid flowing in ‘parallel layers’. Due to some friction on the side of the tubing, the flow ultimately develops a parabolic shape, as shown below.

Direction of flow in a cytometer

Into the center of this laminar flow, the cells are injected. The cell and fluid mixture is introduced at a higher differential pressure, keeping the cells in the center of the laminar flow, and allowing the process of hydrodynamic focusing to occur, which causes the cells to spread out along the velocity axis, single file, as they approach the intercept point.

Differential flow within the cytometer during testing

After the intercept, the cells either flow into the waste or, in the case of a cell sorter, the stream is broken into droplets, and the appropriate droplet is charged a ...

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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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