3 – Data analysis

With the increased development of fluorescently conjugated monoclonal antibodies came more applications with potential clinical impact. In bone marrow transplantation, studies using hematopoietic cytokines made it feasible to gather stem cells from peripheral blood. It was also shown that reconstitution of bone marrow was accelerated when using cell from peripheral blood rather than bone marrow. Many more clinical flow cytoemtry applications have been developed. All of which should follow these 6 keys of running clinical flow cytometry experiments.

Flow cytometry data analysis is getting more complex. Gone is the rule of 2-3 color experiments. Even beginners are starting with 5+ color assays, and the adoption of mass cytometry has the potential to increase our headaches even more. Current data analysis methods are good for single tubes or small cohort studies.  What do you do when you have a large dataset, with multiple sampling conditions, and multiple outcome measurements? With data complexity of this nature, one can export the numerical data to a third party analysis package, but even then the analysis can be difficult to perform. To overcome this…

Measuring purity is not the best way to measure flow cytometry cell sorter performance. Recovery is much more sensitive to the correct calculation of a cell sorter's drop delay than the purity. Here's how to calculate cell sorter recovery, yield, and purity, and why you should use recovery over purity to measure cell sorter performance.

Manual compensation is the process of adjusting the compensation based on how the data visually looks. If you have manually compensated data in your lab notebook--strike it out now. Manual compensation results in overcompensated data, yielding incorrect conclusions. If you have issues, explore what those problems are and work to resolve them rather than making up fiction by manual compensation. Here are three keys to automatically compensating your data.

BD Biosciences brand of digital flow cytometers, including the FACSCanto, the LSR-II, FACSAria and Fortessa, utilize a software acquisition program known as FACSDiva. Diva is aptly named as it can be a difficult program to master. However, Diva has come along way over the past 10 years and many improvements have been made to help end-user. Taking time to learn these changes will improve the reproducibility of your data, the chances of your data getting published, and your overall experience on the cytometer. These changes will also save you time. Here are 8 time-saving FACSDiva tips to use the during…

Flow cytometry data are numbers rich. Data from experiments can be population measurements (percent of CD4+ cells, for example), or it can be expression level (median fluorescent expression of CD69 on activated T cells). Many times, researchers are content to show histograms to illustrate their point after a flow experiment. This approach misses the opportunity to take that content rich data and extend the analysis into a statistical analysis. To properly perform statistical analysis, the first step is to understand the hypothesis. The hypothesis will guide the statistical analysis, identifying the correct test to be performed. There are several things…

Cell death is a fact of biological life.  How, when, where and most importantly, why cells die, can have huge biological consequences on the path an organism may take. Apoptosis, or programed cell death, can result in a selective advantage for an organism. Fingers, for example, are the result of apoptosis of cells during development. Next to immunophenotyping, measuring apoptosis using flow cytometry is one of the most common assays. It may be because of the many different ways to measure the process, many of which can be easily performed in a high-throughput manner, or combined with other assays to determine if…

I often have researchers come into the core wanting to look at the activation and downstream signaling events that occur in different immune cells. These events occur in response to signals such as cytokines, chemokines, various receptor ligands, and the engagement of the T cell or B cell receptors. The signaling events are also characterized by the initiation of several phosphorylation events. Measuring Phosphorylation Events When this is the case, I recommend that the researchers set up a phospho-specific flow cytometry, or phospho-flow, experiment. These types of experiments measure the phosphorylation state of intracellular proteins at the single cell level.…

Measuring cell proliferation can be done in a number of ways. There is the below tried and true method of counting cells. This straightforward assay can help determine if the cells are proliferating and by comparing counts. Here, a researcher can determine that the experimental treatment is increasing cell growth. A second method of measuring proliferation involves using a radioactive tracer like 3H-Thymidine. In this assay, the amount of the isotope taken up by the cells correlates to the amount of DNA synthesis, and therefore growth. Of course this requires using radioactivity and all that entails. A third method is…

So you just got the most amazing results of your life and you can wait to show it off in lab meeting, or create the figure for a publication. Here are a couple of tips that will help you ensure that everyone else also sees how amazing your data is! 1. If you only have a few events, use the option to “show large dots”. When you only have 4-5 events in a population, it can often be difficult to see. If you turn off the high resolution, you can see the data better. Double click on a plot in…

Friday is the 4th of July in the US – and we celebrate that day with picnics, spending time OUTSIDE the lab and fireworks. And our Independence, but I’m not up for a political discussion right now. For flow geeks, fireworks are like flow cytometry – they happen in the dark, they are full of many bright colors, and we’re all looking for the patterns the colors make. So in honor of a day outside the lab, it seemed appropriate to talk about fluidics… going with the flow for best results. A flow cytometer has three major components – fluidics,…

Fast, accurate or efficient – pick two. How to decide when you can’t have it all. The hemocytometer is considered the gold standard for cell counting. Invented by Louis Charles Malassez, this precision etched microscope slide can allow the researcher to count their cells under the microscope with amazing accuracy. It is inexpensive, relative to other methods, but is by no means the most efficient or fast method out there. The single biggest key to using a hemocytometer is training, training and more training. Since the investigator is visually inspecting the cells within a boundary, the rules of what cells…