4 – Experimental Design

Experimental Controls For Reproducible Flow Cytometry Measurements

By: Tim Bushnell, PhD

With the increased focus on reproducibility of scientific data, it is important to look at how data is interpreted. To assist in data interpretation, the scientific method requires that controls are built into the experimental workflow. These controls are essential to minimize the effects of variables in the experiment so that changes caused by the independent variable can be properly elucidated. Getting into the mindset to improve the reproducibility of flow cytometry experiments requires a hard look at the appropriate controls to use in each experiment.

Procedural Limitations That Impact The Quality Of Rare Event Flow Cytometry

By: Tim Bushnell, PhD

Stem cells, circulating tumor cells, and minimal residual disease in cancer patients were all discovered through the power of rare event flow cytometry. When preparing for rare event analysis, sample preparation and data analysis must be taken into account at the beginning. How will we stain our cells? How will we analyze our cells? What controls will we use to help us identify our rare events? What statistical methods do we use to analyze our results? Here are 5 procedural limitations that impact the quality of rare event flow cytometry data and how to optimize your assay to get the…

5 Best Practices For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

Here, we cover 5 lessons from the trenches of flow cytometry looking at important aspects of how best practices have changed over time, which practices need to be adopted, and which are outdated. Put those old, coffee-stained protocols away and take advantage of the best practices for digital instruments to write new and improved ones (coffee stains optional). Your data will thank you.

Planning For Surface Staining Of Cells In Flow Cytometry

By: Tim Bushnell, PhD

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 to move from sample to cytometer, 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 what other factors should be considered when staining cells? This article will focus on staining immune cells, but the principles apply in general,…

4 Ways To Achieve Reproducible Flow Cytometry Results

By: Tim Bushnell, PhD

There are several areas that researchers can focus on to improve the reproducibility of their flow cytometry experiments. From instrument quality control, through validation of reagents, to reporting out the findings, a little effort will go a long way to ensure that flow cytometry data is robust, reproducible, and accurately reported to the greater scientific community. Initiatives by ISAC have further offered additional levels of standards to support these initiatives, which were developed even before the Reproducibility Crisis came to a head in both scientific and popular literature.

3 Guidelines For Setting Compensation Controls In Flow Cytometry Experiments

By: Tim Bushnell, PhD

Fluorescence compensation is not possible without proper controls, so it is critical to spend the time and effort to generate high-quality controls in the preparation of an experiment. For a compensation control to be considered “good” or “proper”, each compensation control must be as bright as or brighter than the experimental stain, autofluorescence should be the same for the positive and negative populations used for the compensation calculation in each channel, and the fluorophore used must be the exact fluorophore (i.e. same molecular structure) that is used in the experimental sample.

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

By: Tim Bushnell, PhD

The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.

Strengths And Weaknesses Of Isotype Controls In Flow Cytometry

By: Tim Bushnell, PhD

While controls are critical for minimizing the effects of variables in your flow cytometry experiments, choosing the right controls are essential. When your research is published, reviewers need to see that your variables have been analyzed properly. Evaluating strengths and weaknesses will give you information and back up arguments for the case for or against isotype controls. Here’s a review of what isotype controls are and if you need to use them.

How To Prepare For A Flow Cytometry Experiment

By: Tim Bushnell, PhD

Flow cytometry is a very powerful tool and can answer many questions if the experiments are properly designed. There is a learning curve that takes a bit of time, patience, and practice, but soon you may be finding excuses to perform flow cytometry experiments and we will be here to help you with best practices. Using this checklist will help you to design and perform consistent experiments every time.