Cytotoxicity – Moving Away from Biochemical Endpoints
Author: Christopher Black, Ph.D.; Published: Nov 19, 2010; Category: Cytotoxicity, Flow Cytometry; Tags: Flow Cytometry, Toxicity; No Comments
Efforts are currently underway by many groups in the pharmaceutical industry to move from historical observation-based toxicity approaches to more predictive, mechanism-based approaches (e.g. EPA’s ToxCast™, the US National Toxicology Program, and National Center for Toxicogenomics).
In order to accomplish this, models and tools are required that allow for characterization of critical toxicity pathways such as common signaling pathways (e.g. p53, Nrf2), apoptotic pathways, etc. Much is already known about these common toxicity pathways and can be exploited through the use of commercial kits and published assays. However, in order to build a predictive model, more than just endpoints and assays are required—context needs to be given.
Context can relate to the organ that is being modeled (e.g. liver, kidneys), the whole organism (e.g. human, mouse), or even the compound that is being assessed (e.g. metabolism, interactions). The definition, and ultimately the models and tools, need to be defined up front in order to make successful predictions. Inherently, the difficulty is in both choosing the right systems and also in putting together the information in a way that is meaningful to the original question.
Given that there are many fairly straight-forward fluorescent based assays (e.g. Live/Dead®, Mito-Tracker®) which can be applied to the problem of toxicity testing, there is a lack of integration between these endpoints in assembling a clear picture of cell function and biochemistry.
What is sorely needed is a baseline using relevant cell types that helps to establish the “typical” function of a cell system under a defined set of conditions. Key to establishing this suite of baseline assays is the use of pathway-specific positive controls. Once this is established, perturbations can be made and compared against this baseline.
While many kit assays are available and fairly easy, they are typically used for quick checks of relevant endpoints, or to look at acute effects similar to a maximum tolerated dose (MTD). Many times these single readouts are then used to predict cytotoxic behavior. For example, there is ample evidence that cells that are undergoing necrosis will upregulate many other death pathways and processes, including enzymes such as caspases and release activators like cytochrome C.
With a single-point averaged readout, how is it possible to assemble the correct picture of toxicity? Bringing together multiple endpoints along key pathways is the next frontier as it relates to cell-based assay work.
Well Populations and Flow – the Future?
Author: Christopher Black, Ph.D.; Published: Nov 19, 2010; Category: Flow Cytometry; Tags: Apoptosis, Cellular Pathways, Flow Cytometry; No Comments
Flow cytometry has long been used as a powerful cell analysis tool to examine complex populations such as immune cell distributions in the blood stream. With the ability to perform screens using flow cytometry, another interesting possibility exists in the application of this technique for drug screening. Specifically, given a single well in a multi-well plate, we can probe what the cell population distribution is in that well based on the responses of those cells.
Consider the treatment of a single cell line, such as Jurkat cells, with a chemical substance known to be toxic. Even at no concentrations of the chemical there will be cells that are in their death spirals among many healthy and viable cells. In other words, no cell population is completely homogenous. Increasing the incubation time as well as dose will cause more of these cells to enter these death spirals.
However, in every case where some cells exist, there are subpopulations of cells that are completely viable and intact. Any quadrant analysis will easily highlight this distribution. In fact, in this toxicity example, these cells may not have any upregulated stress pathways.
What then is it about these cells that allow them to be non-responders? Measurements on standard plate readers do not allow for such a detailed understanding of cell population –in short, plate readers provide a total overall or summed response of a population.
Since each cell within a population may respond slightly differently to its environment, an understanding of these differences is crucial to building a new molecular approach. A technique like flow cytometry, with single cell resolution, can provide a better mechanistic understanding of how a compound or chemical affects individual cells.
For too long the industry has focused on isolated biochemical endpoints, ignoring the fact that a so-called cell line is not homogeneous and contains information that may be pertinent to the development of more effective, and safer, drugs.
Let’s take advantage of the fact that we can use whole cell models and powerful techniques like flow cytometry to probe these questions. The promise of analyzing interconnected pathways may be right around the corner.
Increased Support and Communication with HyperCyt Users
Author: Linda Trinkle; Published: Nov 18, 2010; Category: Flow Cytometry, HyperCyt User Support; Tags: Flow Cytometry, HyperCyt User Group; One Comment
Yes, it is official—we have created the HyperCyt Users Group and a new vehicle for communication—this blog, which we’ve dubbed “HyperBlog.” Our first HyperCyt User Group Meeting will be in San Francisco January 10, 2011.
The idea for the HyperCyt Users Group was initiated a couple of years ago over some beers after a workshop in Boston. Somehow that’s not surprising—great city with a great tradition of ideas and pubs. That was to be, (and now is) the B-HUG. We are making plans for the B-HUG meeting will also be in January.
It was appropriate that one of the inventors of HyperCyt, Bruce Edwards, was there that day. In our workshop at Beth Israel, Bruce introduced HyperCyt technology and described how they were actually performing primary screening of small molecules in the Molecular Libraries Screening Center lab in the University of New Mexico’s Cancer Research Center. Cell and Bead-based screening in 384-well microplates! And fast!
It was (and still is) amazing to realize that they could screen millions of compounds using flow cytometry and in volumes of 15µl and less. The UNM Center for Molecular Discovery published assays cover a range of specimens and targets from bacteria to prostate cancer cell lines, and Quorum sensing to GPCR’s.
So over the past couple of years, more and more researchers are connecting HyperCyts to their flow cytometers or implementing our HTFC Screening System, and discovering more and more applications and challenges for us at IntelliCyt to address. With people running everything from yeast libraries to antibody screening or multiplexed apoptosis assays, we get involved in some pretty interesting stuff!
When we decided to update our website earlier this year, we thought about ways that we could get information out to you, our customers, and get information back that could help us deliver the products that you really need, and a blog makes it easy for us to get information out. So, the HyperBlog for HyperCyt users is up and running. Please subscribe to our RSS feed, and stop by and leave comments to our posts.
There are some great websites in our field that provide a wealth of information. For example, the Purdue Bulletin Board has long been a great resource for general flow cytometry users and recently, Beckman Coulter placed the flow cytometry bible on their website—Howard Shapiro’s Practical Flow Cytometry.
What we propose here is to address the more specific challenges relating to creating robust, reliable screening applications, or simply running suspension samples in microplates using HyperCyt technology.
I look forward to sharing information among all of you brilliant and creative HyperCyt users.