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Strengths And Weaknesses Of Isotype Controls In Flow Cytometry

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Using isotype controls in flow cytometry

Written by Tim Bushnell, Ph.D

Controls are critical for minimizing the effects of the variables in a scientific experiment so that the effect of the independent variable can be accurately measured.

In flow cytometry, there are a host of important controls necessary to properly interpret data generated in these experiments. Some of these controls include compensation, fluorescence minus one (FMO), stimulated and unstimulated, reference, and controls.

When it becomes time to publish, the proper use of these controls is critical in convincing the reviewer and reader that the data has been properly analyzed.

The isotype control is an experimental control where a sample is stained with an irrelevant antibody with the same isotype as the target antibody. Cells are gated and positivity is set based on the background staining of this isotype control.

The use of the isotype control to set negativity remains a topic of discussion and can confuse the novice to flow cytometry, especially when a reviewer may request why these controls were not included in a submitted paper.
Overall, the isotype Read More



How Flow Cytometry Converts Photons To Digital Data

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Flow Cytometry conversion of photons to electrons

Written by Tim Bushnell, Ph.D

How exactly does a photon of light become an electron, and eventually a number in the FCS listmode file?

In flow cytometry, cells are labeled with fluorescent tags, passed by an excitation light source, and the resulting emitted photons are collected to become the data that identifies characteristics about the cells in question.

These characteristics can include what proteins are expressed, the cell cycle state, the phosphorylation state of a protein, the calcium state of the cells, levels of RNA expression, and so much more.

If there is a fluorescent probe that can measure a specific characteristic, it can often be used in flow cytometry.

A great resource for reference is the Molecular Probes Handbook, as it contains a wealth of information about different fluorescent reagents that can measure everything from apoptosis to Zn++ ion concentration.

This process involves the detection system and the electronics, and basically following a bouncing photon to its ultimate digitization.

The role of the detector on a flow cytometer is to capture photons and co ...

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How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

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Flow Cytometry measuring apoptosis, necrosis, and autophagy

Written by Tim Bushnell, Ph.D

“If one approaches a problem with order and method there should be no difficulty in solving it — none whatever.”

— Hercule Poirot, Death in the Clouds

Murder is a common theme in the mystery suspense genre. The detectives who solve these murders use a combination of observation and deduction to identify the guilty party. This metaphor suits measuring cell death.

In biology, there are four major pathways for cell death.

The study of the different ways cells die has become known as Cell Necrobiology, as coined by Darzynkiewicz and coworkers in their 1997 review article.

Flow cytometry is ideally suited as a tool to study Cell Necrobiology and, with its plethora of reagents, it is even possible to follow the different steps in these processes.

The four major ways a cell can die are:

Cell death is so important, that it has been the center of several Nobel prizes including ...

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How To Prepare For A Flow Cytometry Experiment

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Preparation is vital before starting a flow cytometry experiment

Written by Tim Bushnell, Ph.D

You never forget your first experiment.

Usually, you remember all the things that went wrong that you need to correct for future experiments.

In flow cytometry, there are so many things that you need to remember to bring to the first experiment, that having a checklist make sense. This checklist is divided into three phases — before the experiment, at the instrument, and after acquisition — with steps listed for each to help you perform your first flow cytometry experiment with ease.

1. Preparation before the Flow Cytometry experiment.

Before embarking on the first flow experiment, there are several things that you should do to become comfortable with the experimental plan. The first is to understand the protocol that will be used to stain the cells.

    1. Meet with the flow cytometry staff. One of the first steps to take before planning your first experiment is to meet with the team that manages the flow cytometer you will be using. These people are a great reference on the ins and outs of flow cytometry experimental design, execution, and analysis. Y
    ...
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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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