3 Reagents For Identifying Live, Dead, And Apoptotic Cells By Flow Cytometry
Written by James McCracken, Ph.D.
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 sample will not divide, your culture will take extra time to reach the needed level of confluence, ruining your experimental timeline and weekend plans. Or maybe you’re sorting cells for a downstream functional assay, stimulating sorted T cells with antigen and measuring production of IFN. If you failed to remove your dead cells first, you could end up with different percentages of dead cells in one sorted sample versus another sample. This means you will end up with fewer vital cells in one set of wells versus another, altering your results ...Read More
What Is Photon Counting And How To Use 8-Peak Rainbow Beads
Written by Michael Kissner
For decades, flow cytometry professionals have been using 8-peak quality control beads to quickly check PMT sensitivity, resolution, and linearity on their flow cytometry and cell sorting systems.
It’s only recently that these beads have become respected not only as a diagnostic tool but also as a flow cytometry educational tool.
As scientists have continued to learn more about the physics of photon collection and conversion to electrons, they have started to realize that 8-peak beads can teach a very compelling lesson.
The lesson involves photoelectron generation and the inherent error in this process that fundamentally governs all flow cytometric measurements.
Photoelectron is the term we used to describe an electron that was generated at the photocathode of a flow cytometer’s photomultiplier tubes (PMTs) as a result of the process that converts a photon to an electron.
What Are 8-Peak Rainbow Beads?
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 amo ...Read More
Why You Need To Use FMO Controls For All Multicolor Flow Cytometry Experiments
Written by James McCracken, Ph.D.
What are the three most important parts of any flow cytometry experiment?
Controls, controls, controls.
Of course, this is mostly in jest. Mostly.
Reagent selection, well-maintained instruments, and other factors are also important. However, you must control for these factors.
Setting up the right controls is critical to determining how your cells are responding to treatment. It’s also important for correctly interpreting your data and drawing correct conclusions. Without the proper controls, you would not be able to compensate your flow cytometry experiments correctly or identify your cells of interest correctly.
By using the wrong controls, or leaving out certain controls altogether, you leave yourself wide open for criticism. Your gate placement will be questioned, your samples will be questioned, and your papers and grants will be questioned.
Why Fluorescence Minus One, Or FMO Controls, Are Important
We’ve all had experience working with someone who wanted to see the “real” samples, not the control samples, first.Read More
How Droplets Are Charged And Drop Delays Are Determined During An Electrostatic Cell Sorting Experiment
Written by Michael Kissner.
They way that droplets are charged is one of the more counterintuitive and complex aspects of cell sorting.
My own experience as a past fledgling cytometrist certainly echoes this assertion. I distinctly remember my struggle to understand how exactly all of the sorting components coalesced to accomplish the instrument’s tasks, but when I finally did, I gained a much deeper understanding of cell sorting as a holistic process rather than a vague sum of its parts.
It’s my hope here to be able to facilitate a more accurate conception of the process, grounded in a reality of physics and mechanics rather than one shrouded in magic.
Towards this end, the most crucial point is that the technique of electrostatic cell sorting, which is the most widely employed type of sorting, is fundamentally built upon droplet charging.
How Flow Cytometry Electrostatic Cell Sorting Works
Electrostatic cell sorting is a complicated process that continues to be improved.Read More
How To Use Flow Cytometry To Correctly Define T Cell Subsets And Their Functions
Written by Jennifer Snyder-Cappione, Ph.D.
Flow Cytometry is a remarkably powerful tool for the study of T cells. It has been successfully used for many decades to accurately visualize and enumerate a variety of T cell subsets.
With a large sensitivity range for fluorescent probes, >95% sampling efficiency, and the ability to sort populations of interest for further study, fluorescent-based cytometry remains a tool of choice for T cell analysis.
Single cell visualization of T cells in a heterogeneous sample is clearest when the defined T cell populations are determined with ‘rock-solid’ gating and data analysis strategies.
For example, detection of the total CD4 and CD8 T cell compartments (via CD3+ CD4+ and CD3+ CD8+ cells, respectively) is straightforward; also, T cell populations that are clearly defined by surface antigen expression include antigen-specific (tetramer-binding) memory T cell clones and invariant Natural Killer T (iNKT) cells, a unique T cell subset discerned via binding to a CD1d-glycolipid loaded tetramer.
Such gating strategies, when paired with CD3 inclus ...Read More