Friday, March 30, 2018

BTB# 13: Dye dilution method for understanding cell proliferation

Cell proliferation assays, are now very commmonly used in a variety of research experiments. With techniques becoming more and more accurate, gone are the days where we use a simple counting of cells to understand the cellular proliferation. On a very broad sense, cellular proliferation assays are of various types including- Cell counting, Cell cycle measurement, BrdU/EdU uptake method, Dye dilution method. Of all these, in this post I will discuss about Dye dilution method since they provide great and easy measurements.

In dye dilution method, cells uptake a fluorescent dye which is stably sitting inside the cell. In an ideal scenario, they are neither metabolised or removed from the cell. When the cell divides, the fluorescent dye is distributed equally between the two daughter cells. This can be captured as a fluorescent signal in flow cytometry. On a best day, this method can trace 5-7 generations of cell divisions. How do you apply this? Let us say, for example your experiment involves understanding what happens to a B cell after 4 rounds of replication, after you have stimulated them. For that you need to first know that the B cell has undergone 4 rounds of division.

Fig 1: Theoretical proliferation analysis. Source
In Figure 1, the parent cell is in generation 0 (G0), which has taken up the dye fully (In practice, the exact concentraion of dye depends on the total dye used and concentration of cell in the mix). When the cell divides, the daughter cell (generation 1) has half the concentration of dyes. The 3rd generation of cell has 1/4th of the G0 and so on. This is reflected in Flow cytometry data, reduced intensity indicates subsequent generation.

Fig 2: Typical plot using
CellTrace Violet. Source
In an example (Fig 2) where cells have been stimulated and left for 48 hours before analysis, shows the histogram representation of data analysis. The plot shows that cells have made a maximum of 4 divisions (shown in red). The unfilled histogram shows the starting population of undivided cells. Note that the 1st generation has higher concentration of dye than the 2nd and hence is more brighter (hence the peak towards right). The height of the peak represents number of cells.

There are several such dyes available in the market. Some of the most common dyes that you might have heard about include CellVue Claret, Cell Trace Violet, eFlour etc. The choice of dye is based on several parameters such as if It is taken up by live cells, Intensity of staining, cellular retention, segregation between daughter cells.

There are two major classes of these dyes, based on where the dye is retained by the cells. 

1. Intracellular dyes
2. Cell membrane dyes (Also known as lipophilic dyes)

Table 1: Common Cell Proliferation Dyes. Source
Intracellular proliferation dyes as the name suggest binds to intracellular contents. One of the most commonly used dye under this banner is the CFDA-SE (Carboxyfluorescein diacetate succinimidyl ester). CFDA-SE enters the cell where it is acted upon by cellular esterases which cleave the compound into CFSE (Fluoerescent compound) which stays intracellularly by binding to intracellular lysine residues and other amine sources. However, the CFSE has a toxic activity and hence these days, Cell Trace Violet is more commonly used. Cell membrane dyes are the ones, which bind to cell membrane contents and give a fluoerescent reading. CellVue Claret, a red dye is one of the best example in this category. Table 1 gives the most Common Cell Proliferation Dyes that are in current use.

Most recently, a method called as HCS (High content screening) is begining to turn out to be more sensitive when compared with dye method. The reason being they are more sensitive and superb signal-to-noise ratios than other methods, owing to their automated measurement of synthesis of new DNA. In reality this system is an improved version of BrdU/EdU uptake method using an automated platform.

Tuesday, March 20, 2018

Disease X: The term is very misleading


It has been nearly more than 5 months since I last wrote a blog piece here all thanks to a busy schedule. There have been too many things that I wish I would have written about. Anyways, thanks to all those readers who enquired if I would still be writing or I have quit. Nope I havent quit. I just took a Mega break.

The latest sensation in the field of infectious disease is something called as "Disease X". This fancy and scary term is talked about in virtually every leading news outlet. After witnessing some of the most discussed Global emergencies such as SARS, MERS and Ebola Disease X is being projected as the next deadly pandemic in the making. So what exactly is Disease X? In its most essential sense, Disease X is a hypothetical infectious agent that is expected to cause a devastating outbreak at a global scale.

According to the WHO, "Represents the knowledge that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease". Dr. Anthony Fauci, further clarfies, "As experience has taught us more often than not the thing that is gonna hit us is something that we did not anticipate. Just the way we didn't anticipate Zika, we didn't think there would be an Ebola that would hit cities." Source
Photo 1: Disease X.

In May 2015, the WHO was requested to come up with a plan for identifing potential pathogens of pandemic capabilities and how it could be handled and tackled. The first list tentative list was published on 10th Decembe 2015 based on expert opinions and understanding. This document formed the basis for "List of Blueprint priority diseases". The orignial list of disease priorities included Crimean Congo haemorrhagic fever, Ebola virus disease and Marburg, Lassa fever, MERS and SARS coronavirus diseases, Nipah and Rift Valley fever.

In February 2015, WHO published a objective methodology called as "Methodology for Prioritizing Severe Emerging Diseases for Research and Development", which is aimed at determining what agents should be classified as priority. This is mainly based on factors such as research and understanding already available, pandemic potential, immunity etc. Based on the assessment, a second revision of List of Blueprint priority diseases was released. The agents include
  • Crimean-Congo haemorrhagic fever (CCHF)
  • Ebola virus disease and Marburg virus disease
  • Lassa fever
  • Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS)
  • Nipah and henipaviral diseases
  • Rift Valley fever (RVF)
  • Zika
  • Disease X
It should be noted that this list is not absolute and is more of a guideline representing where research has to be prioritsed for epidemic/pandemic preparedness. Disease X is an unknown entity. This could be anything that we havent identified yet to cause devastating infection.

Disease X is thus a term given to known unknow that could potentially cause significant problem. In other words, "We have no idea whats on the next in the list". Take into consideration, WHO committee science adviser John-Arne Rottingen says, “It may seem strange to be adding an ‘X,’ but the point is to make sure we prepare and plan flexibly in terms of vaccines and diagnostic tests. We want to see ‘plug and play’ platforms developed which will work for any, or a wide number of diseases; systems that will allow us to create countermeasures at speed.”