2 – Reagents

How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment

By: Tim Bushnell, PhD

When setting up a cell sorting experiment, there are many things to consider. You must consider which controls you’re going to use, how you’re going to compensate the experiment, which instrument and which instrument settings are ideal, and how you plan to analyze, gate, and present your data. With so many things to consider, it’s easy to lose site of the small things that can drastically affect the viability of your cells, including the composition of your suspension buffer. The composition of the suspension buffer for preparation, staining, analyzing and sorting is perhaps the most important parameter for maintaining viability…

3 Reagents For Identifying Live, Dead, And Apoptotic Cells By Flow Cytometry

By: Tim Bushnell, PhD

There are several methods for analyzing live, dead, and apoptotic cells by flow cytometry. As cells die, the membrane becomes permeable. This allows for antibodies to penetrate the cells, which can now mimic live cells. For this and other reasons, it’s important to remove dead cells from further analysis during your flow cytometry experiments. For example, let’s say you merely need to generate an accurate cell count. If you fail to remove your dead cells first, you might think you’re seeding 10,000 cells, but in reality only 7,000 of your cells are actually viable. Since the dead cells in your…

What Is Photon Counting And How To Use 8-Peak Rainbow Beads

By: Tim Bushnell, PhD

8-peak beads, sometimes called “rainbow” beads, are a set of beads in a single vial that contains 8 different populations that differ only in the amount of fluorophore contained within them. One of the peaks, termed Peak 1, is unlabeled, and the additional seven, termed Peaks 2-8, contain increasing amount of fluorophore. 8-peak beads are designed to fluoresce in all channels on most flow cytometers and cell sorters. These beads are used to check fluorescence sensitivity and resolution by measuring the position of the unlabeled peak and the separation between all of the peaks, respectively. They are also used to…

Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments

By: Tim Bushnell, PhD

FMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them. When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. It’s a strong negative control because the left out marker in the FMO control allows you to take into account how the other stains in your panel affect the respective minus parameter. Many flow cytometry gates are difficult to define. This is especially true when you’re looking at activation markers within a continuum or accounting for the large…

7 Advanced Flow Cytometry Data Analysis Tips For Multi-Color Experiments

By: Tim Bushnell, PhD

In today’s world, many scientists have access to instruments capable of running experiments with 10 or more colors. The leap from 2 to 10 colors may seem small, but here are many factors to consider in the design and analysis of experiments that makes full use of instruments that can handle these additional colors. Imagine analyzing a 2-color experiment. With 2 biaxial plots and a single quadrant gate, you have only 4 populations to report. Now add a 3rd color. By doing so, you’ve increased your population count to 8. With 4-colors, you’ve increased your population count to 16. On…

Why Understanding The Jablonski Diagram Will Help You Publish Your Flow Cytometry Data

By: Tim Bushnell, PhD

Flow cytometrists use the Jablonski diagram to aid in understanding and explaining the kinetic events of fluorescence. Fluorescent compounds start at the ground state until they are excited by interacting with a photon of light. This photon excites the compound, promoting an electon to a higher energy state. Some of this energy is lost by emission of heat and other non-radiative processes, leading to the previous energy state. Finally, an electron falls back to the ground state while releasing a photon of light. This photon has a lower energy (higher wavelength) than the exciting photon of light. Here's how understanding…

When To Use (And Not Use) Flow Cytometry Isotype Controls

By: Tim Bushnell, PhD

The field of flow cytometry is moving beyond the use of isotype controls, with many suggesting they be left out of nearly all experiments. Yet, isotype controls were once considered the only negative controls you should ever use. They are still very often included by some labs, almost abandoned by others, and a subject of confusion for many beginners. What are they, why and when do I need them? Are they of any use at all, or just a waste of money? Most importantly, why do reviewers keep asking for them when they review papers containing flow data? Here is…

How To Create The Right Flow Cytometry Antibody Panel Every Time

By: Tim Bushnell, PhD

Sudoku puzzles seem to be all the rage. I see it in coffeehouses, at the airport, even in doctors offices. Everyone is trying to work out how to fit the numbers into the grids so that everything adds up properly. Designing polychromatic flow cytometry panels is much like the Sudoku puzzle. In this case, the grid is composed of the antigens on one side, and the cytometer detectors on the other. The goal is to fill in the grid correctly.   Instead of adding up to 45, like in Sudoku, the flow cytometrist is trying to optimize the ability to…

The Most Common Mistake Researchers Make When Designing Flow Cytometry Antibody Panels

By: Tim Bushnell, PhD

Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help. With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors. Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation (Figure…

If You Don't Know This About GFP, FITC, And PE, You Might Publish False Flow Cytometry Data

By: Tim Bushnell, PhD

When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb. What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the…

How To Design Accurate & Effective Flow Antibody Panels (or, What's An OMIP?)

By: Tim Bushnell, PhD

I was at a meeting talking about the principles of panel design. At the end of my talk, I had an investigator approach me and ask why he was not making progress on his 15-color panel that he started developing. So, I asked how long he’d been working on it. A month. That was his response. This might shock some of you but a month is not very long when it comes to designing an accurate and effective antibody panel for a flow cytometry experiment. Multicolor panel design requires a delicate balance of biology and physics. Understanding the biology of…