Fluorescence compensation is not possible without proper controls, so it is critical to spend the time and effort to generate high-quality controls in the preparation of an experiment. For a compensation control to be considered “good” or “proper”, each compensation control must be as bright as or brighter than the experimental stain, autofluorescence should be the same for the positive and negative populations used for the compensation calculation in each channel, and the fluorophore used must be the exact fluorophore (i.e. same molecular structure) that is used in the experimental sample.
Getting a clear signal with reduced noise is an essential component to good data. Adding a threshold when acquiring flow cytometry data is one way to do that. It reduces the number of events by setting a bar that a signal pulse must clear before it is counted as an event. Depending on the importance of the data, the downstream applications for the data (or sorted cells) will dictate how critical the threshold is. In combination with proper sample preparation, appropriate thresholding will reduce debris and ensure best outcome.
The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.
While controls are critical for minimizing the effects of variables in your flow cytometry experiments, choosing the right controls are essential. When your research is published, reviewers need to see that your variables have been analyzed properly. Evaluating strengths and weaknesses will give you information and back up arguments for the case for or against isotype controls. Here’s a review of what isotype controls are and if you need to use them.
From photon, through conversion from light, into an electron, into an electronic signal pulse. The journey of a photon, through to its data representation via flow cytometry, involves the detection system and the electronics, and basically following a bouncing photon to its ultimate digitization. This article provides 3 key takeaways in the process from photons through to end data for analysis.
Using flow cytometry and a host of different reagents, it is possible to tease out how your cells may have died. Using these tools, you can readily eliminate the various suspects and come to your conclusion as to how your treatment may have killed your cells of interest. Here are some reagents to consider when measuring apoptosis, necrosis, and autophagy.
Flow cytometry is a very powerful tool and can answer many questions if the experiments are properly designed. There is a learning curve that takes a bit of time, patience, and practice, but soon you may be finding excuses to perform flow cytometry experiments and we will be here to help you with best practices. Using this checklist will help you to design and perform consistent experiments every time.
T cell differentiation and effector function regulation is an area that needs to be better understood. Until then, speculation combined with best practices help you determine your T cell population’s status. Functional profiling is the primary determinant with surface markers as supporting evidence to assess whether your T cell is activated, resting, or naïve. Here are ways to help differentiate your T cells.
Poorly designed panels, failure to plan for the end-results, and not taking into account instrument characteristics, can all result in a failed sort, lost opportunity, and delay in the necessary data. Take the time at the beginning, before starting a rare event sorting, to understand the different issues that will potentially impact your outcome, and develop a plan to address each of them. Here are 4 considerations to keep in mind.
Most of the interactions that a user has with a flow cytometer is with the fluidics system, and many of the issues that users will face in troubleshooting problems on the instrument will also be here. Understanding how the fluidics system works on your flow cytometer will help you prevent many common issues, prepare your samples correctly, and protect your data. Here are four important things to consider about the fluidics system in a flow cytometer.
Fluorescence compensation is one of the more difficult, understandably confusing, and misunderstood aspects of flow cytometry. Understanding what compensation is, why it is necessary, and what to expect when using it, are critical for generating useful and high-quality data from flow cytometry experiments. The definition and mechanics of flow cytometry compensation and the critical concept that compensation’s most basic principle relies on are discussed here.
Flow cytometry is a powerful technique impacting both clinical and research. When looking for a career, flow cytometry can take you many places. An experienced flow cytometrist can find a job in a biotechnology company, academia, a clinical setting, and more. To be successful in the field, it’s important to seek out new educational opportunities and network with your peers. Here are 5 tips that can help you turn flow cytometry into a successful career.