2 – Reagents

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

By: Tim Bushnell, PhD

Cell cycle seems like such a straightforward assay. At its heart, it is a one-color assay and should be a simple protocol to follow. However, as discussed before, fixation and dye concentrations are critical. Once those are optimized, it becomes important to run the cells low and slow in order to get the best quality histograms for analysis — the topic of another blog. Adding the critical CEN and TEN controls will help standardize the assay, and ensure consistency and reproducibility between runs while helping identify non-standard (aneuploid, polyploid) populations from normal ploidy. Trying to isolate and focus on specific…

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

By: Tim Bushnell, PhD

Cell cycle analysis appears to be deceptively easy in concept, but details are absolutely critical. It is not possible to hide the data if there is poor sample preparation, incorrect dye ratios, too much (or too little) staining time, etc. Forgetting RNAse when using PI will doom your data to failure. Take these basics into account as you move into performing this simple, yet amazingly informative assay.

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

With the added emphasis on reproducibility, it is critical to look at every step where experiments can be improved. No single step makes an experiment more reproducible, rather it is a process, making changes at each stage that leads to reproducibility. Antibodies comprise a critical component that needs to be reviewed. As Bradbury et al. in a commentary in Nature pointed out, the global spending on antibodies is about $1.6 billion a year, and it is estimated about half of that money is spent on “bad” antibodies. This does not include the additional costs of wasted time and effort by…

5 Essential Beads For Flow Cytometry Experiments

By: Tim Bushnell, PhD

Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer. Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control,to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

By: Tim Bushnell, PhD

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 Protocols To Prevent Sample Clumping

By: Tim Bushnell, PhD

Good flow cytometry depends on a high quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze. Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Sample preparation becomes the critical first step in any flow cytometry experiment. To get high quality results, follow these 3 sample preparation steps.

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

By: Tim Bushnell, PhD

Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation. Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’, is not the correct way to determine proper compensation. The best practices for compensation involve following some very specific rules. Here are 4 steps to correctly compensating 4+ color flow cytometry experiments.

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

By: Tim Bushnell, PhD

T regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens. Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells). Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated. This article will show you how to quantify…

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…