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5 Essential Calculations For Accurate Flow Cytometry Results

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Flow cytometry is a numbers game. There are percentages of a population, fluorescence intensity measurements, sample averages, data normalization, and more. Many of these common calculations are useful, but surrounded by misconceptions. This primer will help you decide which calculation to use, when to use it, and how to interpret the results.

1. Staining Index

The staining index (SI) is a way to measure the relative brightness of a fluorochrome and compare it to other fluorochromes in a biologically relevant manner.

The SI is useful for ranking fluorochrome brightness on your instrument of choice. It is also a useful tool for evaluating titration data.

SI is a relative number, so it is best to focus on comparisons, and not the absolute value.

In the case of making a decision as to which fluorochrome is brighter than another, there are sites like this one at Biolegend, and this one at BD, that give a relative rank based on a standard analysis. These are useful, but if your system is significantly different from the standard, you may benefit from performing the experiments your ...

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Measuring Receptor Occupancy With Flow Cytometry

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Written by Tim Bushnell, Ph.D

The field of medical therapeutics is moving into the area of precision medicine. In a global sense, precision medicine requires the doctor to assess a patient’s unique disease state — the susceptibilities and resistances of the disease targets to the arsenal of medicines at the physician’s disposal.

This is leading the push towards devising more nuanced tools, and an understanding of what specific patient characteristics dictate which tools to use.

For precision medicine to work, we must be able to identify biomarkers that are expressed on diseased cells, but absent on the normal cell.

An example of this type of biomarker is overexpression of Her2 on a subset of breast cancers. The drug Herceptin targets the Her2 overexpressed on these cells. Studies suggest that the binding of Herceptin induces an immune response, as well as causing a G1 arrest, reducing cell proliferation.

The success of drugs like Herceptin and Rituximab is one of the reasons there are hundreds of drugs of this class in development.

The ability to perform quantitative a ...

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3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities

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As new instruments come on the market, vendors are quick to provide data proving the systems’ prowess including sensitivity, speed, and such. These are important characteristics of the instrument, and should be reviewed. However, the real questions that should be asked about any new instrument should look beyond these benchmarks. Specifically, the questions that often come to mind include:

  1. Will the new instrument improve current experimental workflows?
  2. Will the new instrument enable new and novel experimental questions?
  3. Will the new instrument help improve the reproducibility of experiments?

Evaluating the instrument in the context of these questions will help determine if acquiring the instrument will expand the capabilities for the local research community. In the case of the ZE5 Cell Analyzer, it is clear that with the advancements that have been made by the Propel and Bio-Rad teams, this instrument offers significant expansion of capacity, resulting in improved reproducibility of the data.

Several features of the ZE5 stand out as prime examples of why this new instrument is ...

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How To Perform A Flow Cytometry t-Test

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Written by Tim Bushnell, Ph.D

The ultimate goal of any experiment is to analyze data and determine whether it supports or disproves a given hypothesis. To do that, scientists turn to statistics.

Statistics is a branch of mathematics dealing with the collection, analysis, interpretation, presentation, and organization of data. In applying statistics to, e.g., a scientific, industrial, or social problem, it is conventional to begin with a statistical population or a statistical model process to be studied.

One of the first important concepts to take from this definition is the idea of a population. An example population might be all the people in the world who have a specific disease.

It is time and cost prohibitive to try to study all of these people, so the scientist must sample a subset of the population, such that this sample represents (as best as possible) the whole population. How big the population is and what fraction is sampled in the experiment contributes to the power of the experiment, a topic for another day.

Figure 1: Relationship of population, sample size, and statis ...

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Planning For Surface Staining Of Cells In Flow Cytometry

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Written by Tim Bushnell, Ph.D

One of the most common assays in flow cytometry is the surface labeling of cells with antibodies. Often termed “immunophenotyping”, it allows the researcher to identify, count, and isolate cells of interest in a mix of input cells. Every lab has their own favorite protocol, handed down from some hallowed, chemical-stained notebook, and followed as exactly as making a souffle.

The real questions are, which of those steps are critical, and (with changes in instruments and theory) what other factors should be considered when staining cells? This article will focus on staining immune cells, but the principles apply in general, and specific issues for a specific sample type can be optimized in a similar way.

Cell Preparation

A protocol usually starts with a list of equipment that is needed. After that, the next important component is obtaining and preparing a sample. A good, single-cell suspension is essential for quality flow cytometry.

The source of your primary tissue will guide you down a path for processing. For liquid samples like blood, bone marro ...

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