Written By: Tim Bushnell, Ph.D.
Isaac Newton was famous for saying “If I have seen further than others, it is by standing upon the shoulders of giants.” Implicit in that statement is that the information that the giants provided was reproducible. In fact, reproducibility is central to the scientific method and as far back as the 10th century, the concept of reproducibility of data was being discussed by Ibn al-Haytham.
In 2011, Prinz et al. published an article that indicated a case study looking at reproducibility by Bayer Healthcare found only 25% of academic studies were reproducible. This was followed up in 2012 by a report from Begley and Ellis that indicated on 11% of 53 landmark oncology studies were able to be replicated. So it seems that while we are trying to see farther, our lens may be out of focus.
Bruce Booth, writing for Forbes, published an article called “Scientific Reproducibility: Begley’s Six Rules” and in this article, he proposed the following 6 rules that should serve as a roadmap in evaluating scientific work, both published and your own work. These ...Read More
The Power Of Flow Cytometry In Cell Sorting
This really captures the essence of the sorting process. It is also a very scary process for the average researcher. After spending hours and hours preparing samples, they are often handed off to an operator who puts them into the cell sorter. With fingers crossed, the researcher hopes the cells are sorted correctly, and there are enough of them to perform the downstream experiments that are the real goal of the process.
Most of the time, things go right, but now and then, they fail, sometimes catastrophically. C ...Read More
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