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

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flow-cytometry-analyze-rare-cells-heterogeneous-samples

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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flow-cytometer-system

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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