Walk into any flow cytometry facility and you will see one or more cell sorters. These devices use the principles of flow cytometry to isolate phenotypically defined cells to a high degree of purity for any of a number of downstream applications. Even single cells can be isolated for cloning and single-cell genomics analysis, a very hot area of research these days.
This was not always the case. Prior to 1965, if a researcher wanted to isolate cells, their only choice was some form of gradient centrifugation, a bulk separation method. There were no real options for anything with more fine control.
That changed when Mack Fulwyler published this paper in which he described an instrument capable of measuring an object’s Coulter volume and isolating the cells based on this volume. The ingenious part of the system was the use of the technology that Richard Sweet had developed for the “ink jet oscillograph”. This first cell sorter is shown in Figure 1.
Figure 1: One of the first cell sorters built by Mack Fulwyler.
Fulwyler demonstrated, using a mixture of mouse and human erythrocyt ...Read More
As discussed previously, cell cycle assays require optimization of fixation and dye concentrations, but that is just the beginning. There are important considerations when performing the assay to ensure high-quality data. Cell cycle experiments are judged by the CV of the G0/G1 peak, and the best way to get a good peak is to run the experiment as slow as possible. Likewise, since the cell cycle assay is run with linear amplification, the PMTs must be monitored and their linearity measured.
Even with those 2 aspects on the machine mastered, there are additional details (like synchronizing the cell culture) that need to be considered. Even more so is the fact that the cell cycle assay lends itself to multiplexing, allowing for more information to be extracted from each sample. Those add-ons to the basic protocol need to explored and optimized as well.
Thus, here are 6 areas of consideration for cell cycle analysis covering these important topics.
1. Run cell cycle analysis low and slow
Acquisition of cell cycle data is not like phenotyping. First, data is acquired with linear amplifica ...Read More
The lifecycle of a cell can be described in stages. In diploid cells, much of the time they exist in a resting state, where a cell does what a cell does — such as, undergo differentiation. In some cases, the cells go into a quiescent state, where the level of RNA is reduced. When the appropriate signals are received, cells begin to bulk up and start to replicate the DNA in preparation for division into 2 daughter cells. After the synthesis phase, the cells enter a second period of rest, where everything is checked before the cells undergo mitosis and produce 2 daughter cells. The cycle repeats itself until the cells die. The cell cycle is usually depicted as shown in Figure 1.
Figure 1: The Cell Cycle. Image from Wikipedia.
While there are many differences in cells at each stage of the cell cycle, one of the most obvious is the amount of DNA that the cell contains. At the G0 and G1 phase, the cells have a normal amount of DNA (2N for a diploid cell). Upon entering the S phase, the DNA concentration begins to increase until it doubles (4N) and the cells reach the second gap (G2) p ...Read More
In most research labs, there exists a notebook that contains the tried and true protocols for lab members to follow. These hallowed, often coffee-stained, pages teach the researchers everything — from how to make media, passage cells, and run restriction digestions, to how to prepare cells for flow cytometry analysis. These protocols are time-honored and tested, so the new researcher doesn’t question the wisdom of the “Protocols Book”.
Unfortunately, these pages are not refreshed with the best practices that have evolved over time as the technology and our understanding has changed and grown. The “truths” in this book are not always right anymore, but the new user doesn’t necessarily know any differently. It is for this reason that there are suboptimal practices that permeate flow cytometry experiments to this day. The last 2 blog articles have discussed the theory and practice of compensation. This blog article will help shine light on some of these historical practices and why they need to be changed.
You can use a universal negative
The idea behind the Universal Ne ...Read More
Why do we have to compensate flow cytometry data?
Newcomers to flow cytometry are often confronted with one of the most confounding issues in flow cytometry. That is, trying to understand the whole idea of “compensation”. It can be explained theoretically, mathematically, by trial and error, or by “take my word for it”. Depending on the audience, a combination of these are used to get the point across.
Simply put, compensation is the mathematical process of correcting the spectral spillover of a fluorochrome into a secondary detector. It relates to the physics of fluorescence. To understand what this means, let’s start with the Jablonski diagram of fluorescence.
Figure 1: Jablonski diagram of fluorescence. Used user creative commons license. Original.
A fluorescent molecule starts at rest, with electrons in the ground state. When a photon of light hits this molecule, it is absorbed (purple line), promoting an electron to a higher energy state. There are a variety of ways that the energy release can happen — we are specifically interested in fluorescence. When a mole ...Read More