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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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3 Flow Cytometry Gates That Will Improve The Accuracy Of Your FACS Data Analysis

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facs data analysis | Expert Cytometry | how to analyze facs data

Written by Tim Bushnell, Ph.D.

Flow cytometry data analysis typically involves a step where a series of gates are defined to identify the population of interest.

The development of robust gating strategies, and the communication of these strategies can be complex. Figures in papers often only tell part of the story. In the world of multicenter studies and the need for improved reproducibility, understanding the specifics of each gating strategy is critical.

As Maecker et al., pointed out in this article, the coefficient of variation due to data analysis went from 20.5% when performed at individual labs, to 4% when performed centrally. With the development of the FlowRepository, investigators can now get access to deposited data and walk through a published analysis with the raw data. This resource will continue to improve the ability to share and communicate the often complex gating strategies.

When training new users on data analysis, there are several different best practices and gating strategies you should incorporate into your analysis. There are also several misconceptions you ...

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What Is Flow Cytometry Light Scatter And How Cell Size And Particle Size Affects It

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scattering of light in flow cytometry | Expert Cytometry | light scatter on particle size

Written by Mike Kissner

Light scatter is unfortunately one of the more misunderstood concepts in cytometry.

When you first learn flow cytometry, you were likely told the misleading phrase, “Forward scatter signal intensity is proportional to cell size, and side scatter signal intensity is proportional to cell granularity.”

The short story here is that, yes, this can be the case when we’re dealing with typical cells (consider blood granulocytes, which are bigger than lymphocytes, have more intense FSC signals; granulocytes are more “granular” than lymphocytes and thus have more intense SSC signals). However, the truth is that light scatter complexity is belied by this perennial but unsatisfactory introductory explanation.

While particle size (particle radius) certainly does influence light scatter signal, its intensity is a function of a combination of factors, including:

  •   wavelength of laser illumination
  •   collection angle, and
  •   refractive index of the particle and flow medium (sheath) 

It turns out that light scatter intensity has a strong dependence on the rela ...

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