Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel
By: Tim Bushnell, PhD
The heart and soul of the flow cytometry experiment is the ‘panel.’ The unique combinations of antibodies, antigens, fluorochromes, and other reagents are central to identifying the cells of interest and extracting the data necessary to answer the question at hand. Designing the right panel for flow cytometry is essential for detecting different modalities. The more parameters that can be interrogated will yield more information about the target cells. Current instruments can measure as many as 40 different parameters simultaneously. This is exciting, as it allows for more complex questions to be studied. Panel design is also valuable for precious samples,…
The 5 Fundamental Methods For Imaging Nucleic Acids
By: Heather Brown-Harding, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
Avoid Flow Cytometry Faux Pas: How To Set Voltage The Right Way
By: Tim Bushnell, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
Designing Microscopy Experiments Related To Infectious Diseases And Antivirals
By: Heather Brown-Harding, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
My 3-Step Panel Validation Pocket Guide
By: Tim Bushnell, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
Easy-To-Forget Flow Fundamentals That Thwart Bad Science
By: Tim Bushnell, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
Important Controls For Your Flow Cytometry Lab
By: Tim Bushnell, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
4 Factors To Improve Flow Cytometry Cell Sorting Speed
By: Tim Bushnell, PhD
There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…
5 Techniques For Dramatic Improvements In Reproducibility
By: Heather Brown-Harding, PhD
It’s not easy to improve reproducibility in your experiments. Image manipulation has become a major problem in science, whether intentional or accidental. This has exploded with the advent of digital imaging and software like Photoshop. There are even mobile applications like Instagram filters that can be used for imaging trickery. It should go without saying that image reuse/manipulation represents profound dishonesty in science – a field intended to uphold the most stringent possible standards of truthful inquiry! But what about studies with a sloppy or stunted capacity for reproduction? These, too, plague science and hinder our ability to seamlessly move…
3 Compensation Mistakes That Will Ruin Your Flow Cytometry Experiments
By: Tim Bushnell, PhD
Compensation is necessary due to the physics of fluorescence. Basically, compensation is the mathematical process of correcting spectral spillover from a fluorochrome into a secondary detector so that it is possible to identify single positive events in the context of a multidimensional panel. Good compensation requires that your controls tightly adhere to three rules. If the controls don’t meet this criteria, it will lead to faulty compensation resulting in false conclusions and poorly reproducible data. Even among flow cytometry veterans, a strong foundation is occasionally in need of a tune-up. And in a topic as complex as flow cytometry, it’s…
Understanding Reproducibility in Flow Cytometry - It’s the Antibodies!
By: Tim Bushnell, PhD
Reproducibility is key to the scientific method. After the results of a study are published, the community validates the findings and extends them. If the findings are not reproducible, the second step is impossible. With performable experiments increasing in complexity, and the concurrent increase in the cost of equipment and reagents to perform these experiments, it is important to find the best way to maximize the money spent on advancing research. In flow cytometry, there are many places where improvements can be made to increase the consistency and reproducibility of an experiment. The most obvious place is in the instrument,…
3 Components Of Every Flow Cytometer You Don’t Know Enough About
By: Tim Bushnell, PhD
All flow cytometer instruments have a certain 3 components, and the way they are put together will dictate the performance of the system. As a user, you’ll be interacting heavily with these components, so you need to know both what they are and how they work. There are fluidics, optics, and electronics. The fluidics allow you to interact at the right flow rate so that your data keep a tight CV. Then you can run the same flow rate for all your samples, and you won't have different CVs for different samples. There are also different optics you can use,…