dry vs wet

Should You Switch from Wet to Dried Blood Samples?

A Spot of History Most of the biomedical methods used started as a curiosity. Then the one-off gains a limited use, the technology then progresses until its use becomes widespread. Just think about the arch from the curious polished glass spheres, used by Antony Levnhook to look at animalcules, to modern microscopes. The same story…

Isolating Monocytes from Whole Blood: A Step-by-Step Guide

Isolating Monocytes from Whole Blood: A Step-by-Step Guide

If you look at the composition of peripheral blood, using hematology microscopy, you’ll see that it’s composed of multiple different cell types, including monocytes. It’s possible to isolate these different components to study and experiment on them directly. So, if you’ve done a few experiments and had fun with THP-1 cells, you can move on…

Guidelines for Efficient Cell Sorting – Part 1

Flow cytometry is a pervasive tool to characterize just about anything in cell biology. From quantifying the expression of surface antigens, to determining the physiological changes in cells and everything in between, flow cytometry is as indispensable to a cell biologist as a knife is to a surgeon. Cell sorting is pivotal in enabling researchers…

How to Perform DNA Extraction from Dried Blood Spots Using Chelex Resin

Every bio- scientist who wants to analyze DNA knows that the process begins with the extraction of DNA from cells of interest. These cells could be RBCs, parasites, or bacteria to name a few. Furthermore, there are various DNA extraction methods1  to choose from depending on sample type, downstream analysis, and so forth. Many scientists…

reagents

How to Store Your Reagents, so They ‘Do Exactly What It Says on the Tin’

Your reagents should do ‘Exactly what they say on the tin.’  This only happens though if you look after them in the way the manufacturer states on their data sheets. We have all been guilty of using reagents past their expiration date.  Usually we can get away with it, but there are a few things…

cytometer

The Difference Between an Image, Flow, Time-lapse and Cell-sorting Cytometer

Ah, cell counting — it’s the oldest trick in the book! Well, not really, but people have been developing methods for counting cells since the late 1800s. It has been around for a while. But what different methodologies are available to biologists now? Well, hold on, because you’re in for a treat! In this article, we…

chromium release assay

Chromium Release Assay: The Old School Way of Testing Cytotoxic T Cells

There are several methods you can use to see if your T cells are cytotoxic, but a chromium release assay using radioactive 51chromium (51Cr) is one of the oldest. It gives good results, and is great for labs that can’t afford or don’t have flow cytometry readily available. Here, I will outline a simple method…

pre-analytical sample

Pre-Analytical Sample Handling: What Can You Do Better?

If you study human disease, you will likely handle a pre-analytical sample or two (or hundreds).  For example, you could handle whole blood, serum or plasma, tissue biopsies, urine, fecal samples, cerebrospinal fluid, or synovial fluid—to name a few. You will probably use these samples to look for specific metabolites, proteins, or nucleic acids that provide…

Demystifying the Flow Cytometry Optics System:  A Peek Under the Hood

Demystifying the Flow Cytometry Optics System: A Peek Under the Hood

To many users, the flow cytometer is a magic box: put in cells, get out data. You click the button to tell it which colors to look at without much thought about how the machine does this. However, not all fluorophores are created equal—some configurations might exclude the spectrum you’re really looking for. Here’s a…

human clinical samples

Four Tips for Working with Human Clinical Samples

While using human clinical samples in your research can provide robust and heterogeneous results applicable to larger portions of the population, working with these samples presents its own set of challenges. Here are some tricks I have learned to help isolate and grow your cells of interest while eliminating stromal, blood, or other undesired contaminants….

fluorescent labels

The History and Future of Fluorescent Labels: We’ve Come a Long Way, Baby!

If you’ve been keeping up with our recent series of articles, welcome back! If not, you can catch up on how fluorescence works or what not to do with your flow experiment. In short, we have been discussing fluorescent labels and their role in flow cytometry. Today, I’ll round out our discussion by touching on…

Corralling Your Cells: How to Gate in Flow Cytometry

Flow cytometry. Some people love it—most hate it—but all can agree that it is one of the most powerful analytical tools immunologists possess. Here’s a quick refresher: as the name suggests, flow cytometry measures the physical and chemical characteristics of cells. This is accomplished by fluorescently labeling cell surface markers/proteins using antibodies conjugated to fluorophores….

fluorophores

Lighting the Way: Understanding Flow Cytometry Fluorophores

As science is becoming more interdisciplinary, the tools we use to answer questions are also crossing party lines. Case in point: flow cytometry. Once a tool only used by “real” immunologists, flow cytometry is fast becoming a method by which numerous questions can be answered, from the length of a cell’s telomeres, to the state…

Analyzing Cell Signaling with Flow Cytometry: Go with the Flow

Phosphorylation Equals Cell Signaling! How do cells communicate and respond to their environmental cues? This question has been on the hot list for scientists ever since the discovery of the cell. Cells use signaling cascades based on biochemical reactions to deliver or receive messages. How cool is that? The major secret of cell signaling was…

flow cytometry data

How to Destroy your Flow Cytometry Data in 3 Easy Steps: Snap, Crackle, and Pop

While many scientists are methodical and precise, some of us like to live on the edge. Read a protocol all the way through? No thanks, I’ll take my chances and guess what concentration of HCl I should use. Label my tubes with the correct content? Puh-lease – it’s much more exciting deducing which is which…

primary murine B cells

The Care and Keeping of Primary Murine B Cells

So you want to work with mouse B cells? Primary murine B cells are a difficult, yet fascinating system to work with and can help deepen your understanding of an immunological system. You can study many things with primary B cells, including: immune activation antibody production cell-cell interactions between immune cells and immune phenotype These…

An Introduction to Spectral Overlap and Compensation Protocols in Flow Cytometry

It strikes fear into the hearts of new cytometrists. Compensation. More fights have started over the proper way to compensate at meetings than anything else. This article will strive to shed some light on the principles of compensation, and equip you with the tools necessary to achieve compensation mastery for your research experiments. Compensation is…

Basic Parameters Measured by a Flow Cytometer: What is Scattered Light and Absolute Fluorescence?

In a previous article, we went over the basic understanding of the inner workings of a flow cytometer. It’s important to grasp the types of measurements that are being made and, perhaps more importantly, what measurements are NOT being made. For simplicity’s sake, we’re going to frame this discussion in terms of a classical flow…

Spot the Difference: 5 Ways to Improve the Presentation of Your Flow Cytometry Data

Take a look at the dotplot below, are you happy with the way it’s presented? Do you think that you could recreate that experiment? If you were a reviewer, would you accept that figure? Sure, it’s flow plot, it shows 3 populations of which two are gated. Read many journals and you will see data…

Catching Greatness: Measuring Cellular Degranulation

One of the key characteristics of cytotoxic cells (i.e. CD8+ T cells, natural killer cells) is the presence of pre-formed cytoplasmic lysosomal granules. These structures house perforin and granzyme; two molecules that are essential for the lysis of target cells. Upon effector cell activation, granules are polarized toward the target cell and the contents are…

Gateway to the Cellular Kingdom: Cell Fixation and Permeabilization Techniques

One of the much sought after question asked by many researchers worldwide is – “What is the gene expression profile of a single cell within a heterogenous pool of cells?” While mass cytometry is the current ‘hot’ methodology for single cell analysis, the good old flow cytometry can help us perform rapid analysis of single…