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4 Critical Components In Cellular Proliferation Measurement

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cellular proliferation measurement | Expert Cytometry | measuring cellular proliferation

Written by Tim Bushnell, PhD

Cell proliferation is a critical component in biological systems.

While normal cell proliferation keeps the body functioning, abnormal proliferation (such as in cancer) can be a target for therapy.  

Measuring how cells proliferate in response to a stimulus is a time-honored assay in science. This can be as simple as a cell count between untreated and treated cells. More sophisticated assays can include the use of 3H-thymidine or colorimetric assays (MTT assays).

While these all can measure proliferation, they lack the finesse that flow cytometry can bring to the assay – which allows the phenotypic identification of which cells are actually dividing, as well as allowing for calculations of values such as the precursor frequency, the percentage of cells that have divided, a proliferation index, and more.

Proliferation calls for cells to make a trip around the cell cycle, and there are many ways to measure cell division.

The focus here is on the long-term measure of cell division—the ‘temporal’ dimension for measuring such biological processes ...

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4 Spectral Viewers You Should Be Using For Your Flow Cytometry Experiments

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fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

Written by Tim Bushnell, PhD

At the heart of flow cytometry is the ability to make meaningful measurements of fluorescently tagged cells.

These fluorochromes can be bound to antibodies, a fluorescent protein, a reporter fluorochrome, and the like. Free online spectral viewers are useful in a variety of ways, all of which help improve experimental design and troubleshooting.

These spectral viewers have a special place on every scientist’s browser toolbar. I refer to them regularly, at least weekly.

During the process of panel design, it is useful to have these open to check and compare different fluorochromes. In fact, with recent upgrades to FluoroFinder, there is an integrated spectral viewer based on the filter configuration.

There are a host of different spectral viewers available online. Each one has its strengths and provides specific information. This often necessitates having to use two or three of them to get the information you want. These are the spectral links I use (in alphabetical order):

  1. AffymetrixFluorPlan Spectra Viewer
  2. BD Bioscience Spectrum Viewer
  3. Biolegend Spect
  4. ...
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The Difference Between Purity, Single Cell, And Recovery Cell Sorting Techniques

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cell sorting techniques | Expert Cytometry | cell cytometer

Written by Michael Kissner

If you have experience sorting different kinds of cell types on a droplet sorter, you may have noticed that sorting efficiency often seems closely tied to the cell and sample type.

For instance, you may have experienced a better outcome, such as a higher efficiency and more cells recovered, after sorting lymphocytes versus sorting an adherent cell line. There are some fundamental concepts that underpin this phenomenon, and understanding them will help you perform better cell sorting experiments.

What Is Flow Cytometry Cell Sorting Efficiency?

Sorting efficiency, in fundamental terms, is a real-time measurement, generated by the instrument, of how successfully its sorting system is able to resolve cells that we want to sort (target events) from cells we do NOT want to sort (non-target events).

Note that we are talking about the sorting system’s ability to resolve events here (the droplets) and NOT the electronics system.

Efficiency is calculated with the following equation:cell sorting techniques | Expert Cytometry | cell cytometer

The results of this equation are highly dependent on two aspects of the sort: 1) th ...

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12 Flow Cytometry Terms And Definitions Most Scientists Get Wrong

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Flow Cytometry Terms And Definitions | Expert Cytometry | flow cytometry meanings

Written by Tim Bushnell, Ph.D.

The most important part of executing a flow cytometry experiment correctly is actually understanding what you are doing. This means you must understand the terms and definitions that are critical to the field of flow cytometry.

As a scientist, you should not just place your faith in a specialized technician. You should not blindly agree with the data you see in front of you without you yourself knowing what ‘logicle scaling’ means, what ‘differential pressure’ means, what happens when you change the differential pressure on an instrument, and so on.

You must also be able to communicate your methodologies and results intelligently. This means, for example, knowing the difference between flow cytometry, flow cytometry cell sorting, and FACS analysis: the latter merely being a trademarked term “owned” by a flow cytometry company.

Top 12 Most Commonly Misunderstood Flow Cytometry Terms

To help resolve this confusion, we have worked with our 700+ Mastery Class members to compile a list of the top 12 most commonly unknown or commonly misunderstood ...

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How To Compensate A 4-Color Flow Cytometry Experiment Correctly

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4-Color Flow Cytometry Experiment | Expert Cytometry | multicolor flow cytometry

Written by Tim Bushnell, Ph.D.

Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation.

Before jumping into the best practices for compensation of flow cytometry experiments, it’s good to show what NOT to do when performing compensation.

Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’ (thanks to Joel Sederstrom for the term), is not the correct way to determine proper compensation.

For example, review the following figure, and ask yourself what is the best compensation value? This figure shows FITC on the Y-axis, spilling into the PE channel, on the X-axis…

Figure_1 (1)

 

Without knowing the median fluorescent intensity of the positives in the negative channel, or being able to evaluate the spread of the data, it is impossible to determine which of these above plots display the properly compensated values.

4 Steps To Compensating A 4-Color Experiment

The best practices for compensation involve followin ...

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