5 – Flow Cytometry

How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

By: Tim Bushnell, PhD

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

Optimizing Flow Cytometry Experiments - Part 2 How To Block Samples (Sample Blocking)

By: Tim Bushnell, PhD

In my previous blog on  experimental optimization, we discussed the idea of identifying the best antibody concentration for staining the cells. We did this through a process called titration, which  focuses on finding the best signal-to-noise ratio at the lowest antibody concentration. In this blog we will deal with sample blocking As a reminder, there are two other major binding concerns with antibodies. The first is the specific binding of the Fc fragment of the antibody to the Fc Receptor expressed on some cells. This protein is critical for the process of destroying microbes or other cells that have been…

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

By: Tim Bushnell, PhD

Over the next series of blog posts, we will explore the different aspects of optimizing a polychromatic flow cytometry panel. These steps range from figuring out the best voltage to use, which controls are critical for data interpretation, what quality control tools can be integrated into the assay; how to block cells, and more. This blog will focus on determining the optimal antibody concentration.  As a reminder about the antibody structure, a schematic of an antibody is shown below.  Figure 1: Schematic of an antibody. Figure from Wikipedia. The antibody is composed of two heavy chains and two light chains that…

2020 - A Year Turned Upside Down

By: Tim Bushnell, PhD

What an incredible year 2020 has been. It started off like any other year and bam SARS-CoV-2 (aka COVID 19) entered the equation, bringing chaos and havoc to the world. Things kept changing overnight as new rules and regulations popped up. Masking, quarantine, and flatten the curve became common words in the news. How we met, how we interacted changed almost overnight. Throughout all of this, as we look to 2021, there is hope and optimism. Multiple vaccines have been developed, building on years of research into the SARS-CoV virus, with some approved for human use, and others on the horizon.…

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,…

Tools to Improve Your Panel Design – Determining Antigen Density

By: Tim Bushnell, PhD

When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design. When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell,…

This Is How Full Spectrum Cytometry Works

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…

My Proven 5-Point Fast Track To A Career In Flow

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…

Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

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…

3 Ways Flow Cytometry Can Be Used To Research Bacteria

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…

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…

Discover The Myriad Applications Of Beads In Flow Cytometry

By: Tim Bushnell, PhD

What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”