4 – Experimental Design

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,…

4 Ways To Analyze Tissues By Flow Cytometry

By: Tim Bushnell, PhD

Did you know that tissues can be measured by flow cytometry? Flow cytometry is the measurement of cellular processes at the whole-cell level. This definition is useful because it includes not only flow cytometry, but any technique that measures at the level of the whole cell. Microscopy, for instance, is a great example of cytometry. But, what can be measured by flow cytometry? For one, tissues with lots of cells. When flow cytometry is practiced, the cells are broken up. Therefore, any cellular interactions within the sample are also broken up. This includes tissues, cell-to-cell contacts in tissues, and virtually…

7 Things You Didn’t Know About Imaging Cytometry

By: Tim Bushnell, PhD

It has been said that “a picture is worth a thousand words.” We are visual creatures, and we seek to capture and describe the world around us. Some of the earliest evidence for this comes from very old cave paintings found around the world, like this painting of a horse found in the caves in Lascaux, France. With the development of reliable microscopes, such as those developed by the dutch draper Antonie van Leeuwenhoek, we were able to see what was previously invisible, probing the unseen and learning in great detail how organisms worked. Over time, the field of cytometry…

4 Steps To Implementing a QC Program For Your Flow Cytometry Experiments

By: Tim Bushnell, PhD

Quality control is the hallmark of improving reproducibility. QC programs are designed to help determine when the process in question goes off the expected path. Depending on the deviation from the established acceptance criteria will dictate the level of intervention that needs to occur. This can be as easy as cleaning the instrument and rerunning the QC, or as extreme as removing the data from the final analysis. Since there is documentation as to the deviations, this provides the rationale for excluding data.

5 Steps To Improve Your Flow Cytometry Data Analysis

By: Tim Bushnell, PhD

To get the best flow cytometry data you need to be thinking about all the steps in your experiment to ensure that you have high-quality data to analyze. To improve the quality of your analysis make sure you're adding keywords at the beginning of your experimental setup, develop a quality control program, trust but verify any software wizards, use proper controls, and make sure you extract the correct data.

The Right Way To Read A Flow Cytometry Scientific Paper

By: Tim Bushnell, PhD

Reproducibility is a state of mind. It's not one simple thing that you do that will make all your data more reproducible, it a shift in the way one thinks about and perform experiments. With the emphasis on rigor and reproducibility in science, it's very important that researchers start putting into place everything they can do to help improve the quality and reproducibility of there data. Learn 3 action steps that can be taken to enhance experimental reproducibility.

Avoid Data Loss By Following These Steps To Set Your Flow Cytometry Gates Correctly

By: Tim Bushnell, PhD

The way you set your gates in flow cytometry is a key part of your experiment. You need to know that you can find the populations that you're interested in so you can extract the appropriate data. This is how you can do your secondary or statistical analysis confidently. Learn what gates you should be using and how to define them properly.

3 Ways To Measure Cell Death With Flow Cytometry

By: Tim Bushnell, PhD

Cell death is a natural part of the lifecycle of a cell. In cases of development, it is critical for the shaping of fingers during human development. The processes of ordered cell death, or Apoptosis, are so important that in 2002, Sidney Brenner, Robert Horvitz, and John Sulston received the Nobel Prize in Medicine for their work on understanding this process. There are many different ways to measure cell death and flow cytometry is an ideal tool for this technique. Whether you are just assessing the viability of your cells or you are interested in the exact stage of cell…