Flow Cytometry Protocols To Prevent Sample Clumping
Written By Tim Bushnell, Ph.D.
Good flow cytometry depends on a high-quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze.
Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Thus, sample preparation becomes the critical first step in any flow cytometry experiment.
As Henry Ford once said, “Before everything else, getting ready is the secret of success.”
For the purposes of this article, our attention will be focused on mammalian cells and cell-lines. While other samples can benefit from these tips, samples from the environment, bacterial and yeast samples, and small particles like extracellular vesicles, have their own issues and tricks to consider.
To get high quality results, follow these 3 sample preparation steps…
1. Get the purest sample possible.
Taking the example of working with peripheral blood, one of the first and most important choices to be made when using peripheral blood is the type of ...Read More
How Flow Cytometry Optical System Components Work
Written by Tim Bushnell, Ph.D.
The importance of the optical system of your flow cytometer was established in Part I of this series, noting in particular the benefits of foundational knowledge to gain a comprehensive view of your data, as well as troubleshooting throughout your experiment.
In Part I, the role of lasers was broken down. This follow-up article investigates the lenses, mirrors, and filters in your flow cytometer.
To review these three elements…
- Lasers illuminate the stream with coherent, focused light of specific wavelength (energy) and power. This illumination facilitates the generation of fluorescence signals from cells labeled with fluorophores and light scatter signal from redirected laser light.
- Lenses focus laser light and collect light scatter and fluorescence optical signal and direct this signal to the optical detection path.
- Mirrors are responsible for directing light through the detection path and partitioning it so that fluorescence and scattered light are directed to the appropriate detectors.
- Filters, placed in front of detectors, function to restri ...
What Is A Flow Cytometry Laser And How Flow Cytomtery Optics Function
Written by Tim Bushnell, PhD
The optical system of a flow cytometer consists of an elegant coordination of many components that function concordantly and synchronously to generate the signals that we need to measure in order to shed light on the biology at hand.
Understanding the optical system of a flow cytometer may seem unnecessary for performing a typical experiment, but the more you know about your instrument, the better you will be at understanding nuanced aspects of your data, as well as troubleshooting any potential issues that may arise during an experiment.
A cytometer’s optical system can be broken down into two major parts: A) lasers, and B) lenses, mirrors, and filters.
Lasers and some lenses comprise the excitation optics that generate optical signal, while other lenses, mirrors, and filters form the emission optics which collect optical signal. A brief description of the roles of each of these components is listed below, followed by more detailed descriptions.
Part I of this article will focus on lasers. Look for Part II for the remaining discussion on lenses, mirror ...Read More
4 Gating Controls Your Flow Cytometry Experiment Needs To Improve Reproducibility
Written by Tim Bushnell, PhD
To reproduce reliably in flow cytometry, one must control the gate.
The identification of the target cells of an experiment is the critical first step to performing the secondary analysis that will be used to judge the biological hypothesis and is done by peeling away the layers of cells that do not meet the criteria.
This involves the data reduction method of ‘gating’ with the researcher as gatekeeper, controlling what may pass and what shall not pass, based on the controls designed for the specific experiment.
It is disappointing to realize that in the paper, Maecker et al., the authors evaluated different models for conducting clinical trials and found that individual labs experienced a ~20% CV in the data analysis whereas a central lab showed only a ~4% variance in data analysis.
One of the best ways to improve gating is to ensure the most appropriate controls are identified and collected in the experiment.
How these controls are used to identify the population of interest is also critical to improving this process. There are 4 common gating contr ...Read More
How To Improve Reproducibility Through The Automated Analysis Of Flow Cytometry Data
Written by Ryan Brinkman, Ph.D.
Editor’s Note: Reproducibility continues to be a critical area that all researchers need to be aware of. From the NIH’s focus on reproducibility in grant applications, to a renewed focus by reviewers on the way data has been analyzed and presented, it is imperative that researchers keep up on best practices to ensure they pass these hurdles.
One area that flow cytometry researchers should be focusing on is the emerging changes in the area of automated data analysis. Over the last five years there have been dramatic changes and improvements in these programs and workflows. As Dr. Brinkman discusses below, the automated analysis of flow cytometry data is coming into its own.
Flow cytometry (FCM) datasets that are currently being generated will be two orders of magnitude larger than any that exist today, and new instruments, both flow and mass cytometry, have increased the number of parameters measured for each single cell by 50% (to 30).
Even in 14 dimensional datasets there are 16,384 possible cell populations of interest pre-sample (1). ...Read More