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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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What Is A Statistical Analysis T-Test And How To Perform One Using Flow Cytometry Data

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t-test statistical analysis of flow cytometry data | Expert Cytometry | t-test formula for data analysis

Written by Tim Bushnell, Ph.D.

Designing an antibody panel and running samples on a flow cytometer are not the only steps in a flow cytometry experiment.

After you run your experiment, you have to analyze the data. In particular, you need to perform statistical analyses of the data. This is especially true if you’re hoping to publish your data.

Once all the experiments are concluded and the preliminary analysis of the data performed, you must perform statistical analyses on the data to determine if there is significance in the data.

There are several different statistical tests that can be performed depending on the type of data and the comparisons being made. In the case of either making a comparison against a hypothetical mean, or comparison between two populations, the gold standard test is the Student’s T-Test.

What Is A Statistical T-Test?

The T-Test was developed by chemist William Sealy Gosset, who developed the test while working at the Guinness Brewery as a way to monitor the production of their most famous product.

Since he wasn’t allowed to publish his work direc ...

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Top Flow Cytometry Instrument, Reagent, And Software Trends To Pay Attention To In 2016

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flow cytometry software | Expert Cytometry | flow cytometry instruments

Written by Tim Bushnell, Ph.D.

From instruments, to reagents, to software there are many flow cytometry innovations to pay attention to this year.

These are just a few highlights of the great things coming your way in 2016…

I. Instruments

The mid-range cytometry market is becoming crowded with a host of new instruments on the market.

Of those, we’ve had the chance to try the Novocyte, which is a very easy to use instrument built with the Accuri mindset—fixed voltage PMTs. This, coupled with easy to use software, makes it a breeze to get a researcher up and running.

We noticed some issues in the far red channel of the Novocyte, but we’ve been told this is being resolved.

I’m looking forward to testing out the redesigned Attune NxT instrument from ThermoFisher (previously Invitrogen). The acoustical focusing on this instrument makes it very intriguing for rare event analysis, as you can run faster with less spread of the data.

Moving to the higher end platforms—multidimensional data is very exciting to us. The new Helios (CyTOF III) system, from what we’ve read, is solid. ...

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