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If you are establishing a flow core facility or even setting up just one cytometer, then you need to know a few things before you plan your new lab and start looking at purchasing cytometers: Before You Start 1. Know Your Customer It’s really important to know if you are going to be setting up and…
No matter how we make measurements, there will be variation (a spread of data). Take 100 people and ask them to guess your age and you will get a range of results: some will be too low (excellent!), some too high (not so good!). It is the same with any of our laboratory experiments –…
Once you have been in the field of Flow Cytometry for a bit, the name Howard Shapiro will be familiar to you. However, for those who are new to Cytometry, you might not be aware of Dr Shapiro and his fabulous book ‘Practical Flow Cytometry’. Most Cytometrists have a paper copy of this bible of Flow…
After designing a multicolor flow cytometry panel and securing the necessary cells and reagents, the process of optimization of the panel can begin. The first step in that optimization is titration of your antibodies. In this process, following a standard protocol to be used in the final analysis, you stain a known amount of cells with…
“The greater the power, the more dangerous the abuse.” – Edmund Burke “With great power comes great responsibility.” – Uncle Ben (quoting Voltaire) While it’s unlikely that either of these speakers performed multi-dimensional flow cytometry, it is important to remember these quotes in the context of developing and implementing a good polychromatic flow panel. More fluorochromes are…
T cells can be problematic to characterise because they have a wide variety of subtypes and because of the technical difficulties of studying the membrane-bound T cell receptor, but there are situations where you want to be able to do this such as analysing the degree to which immunological memory has been induced to measuring…
“It is the weight, not numbers of experiments that is to be regarded.” Isaac Newton Read any flow cytometry protocol and somewhere near the beginning will state something to the effect of ‘Place 1 million cells into a tube.’ The question is, faced with that special sample for THE experiment, how do you count cells…
If you are bringing cells to a core to be sorted there are a few things you can do to optimise your sorting and will help the core staff. Here are a few hints: 1) Know the size of your cells Do you know the size of your cells? Not many people do and its one of…
Figuring out what’s what When studying cells and cell subsets (and cell sub-subsets, and so on!!) we need ways to identify and classify every single cell. This will allow us to individually analyse each population and, for example, help to discover their role in health and disease. A principal way we do this is by looking…
The golden rule of flow cytometry, especially cell sorting is: ‘Put good cells in and get good cells out’. When you sort you might not get good cells out and you may not get the numbers you were expecting. In my previous article I touched on a few reasons why your cell numbers might be…
If there are a million cells of interest in your sample and you pass them through a sorter, you might expect to get a million cells back. But you don’t – and here is why. Hardware aborts A hardware abort occurs when an instrument can’t process the information about an event because it is still…
Cell sorters do not operate by magic, even it looks that way. It’s about the application of physics, electronics, fast computers and formation of droplets. Whether you bring your cells to a flow core to be sorted or you sort them yourself, it important to know how the cell sorter works. Cells can be sorted…
“What Have You Done To My Cells??!!!” This cry of pain from researchers, frequently aimed at core facility operators, is heard after receiving incomprehensible data for an invaluable tube of cells. Equally baffling to the trained user of flow cytometric instrumentation is when data emerges that is either unreliable or inconsistent with the known properties…
Ever done a multicolour flow cytometry experiment, run all your controls, done your compensation and then started to analyse your data and realised that you can’t work out where to put your gates? To err is human… When acquiring data on a cytometer, there can be measurement errors due to counting statistics, errors in processing…
No, this is not a call for Geeks to take over the world – just a tiny part of it – the part that ensures success in all experiments or at least a good way to analyze them if they fail. There are all too many entry points for error and variability that can be…
Cytokines, those small proteins that modulate immune cell responses, once translated are normally secreted rapidly out of the cell. So, previously we could only check the levels of cytokines secreted in the supernatant, but we wouldn’t know which cell was producing which cytokine. But what if we had a way to keep the cytokines inside the cell? Then we…
Biosafety is one of those things many scientists don’t take seriously. I would guess, that like politics, there are 40% who believe biosafety is ‘over-emphasized’ and 40% who swear by biosafety. 20% are undecided. Needless to say, I’m on the side of biosafety. And here’s why: “CDC announced today that approximately 75 Atlanta-based staff are…
Flow cytometer and cell sorter manufacturers have invested considerable resources to design instruments that are the “fastest in the ‘hood” either in terms of cells analyzed per second, or in total throughput. The general idea is the faster you can go, the quicker you can identify rare cells, and produce sorted populations containing large numbers…
After a very naïve start in flow cytometry thinking that published protocols will work without fault – needless to say, that did not work out in my favor – I realize now that following a few simple steps can go a long way, and will undoubtedly save you time in the end. So, here are…
What is one of the first things you do when you sit down at the flow cytometer and start looking at your cells? You start drawing polygons and setting gates. To the neophyte the gating process can look a little random – why do you exclude those dots but not these? But gating in flow…
Here are a few handy hints for dealing with your flow cytometry core facility colleagues. They are an amazing resource and are the people with the power to get experiments done.
What if there was a way to take the power and speed of a flow cytometer and couple it with the resolution of a microscope? Imaging flow cytometry does just that! Flow cytometry is a very powerful tool for the complex characterization of cells and cell populations but flow cytometers can also be thought of…
You are sitting in a seminar when you glimpse it: the figure that beats all other figures – a beautiful contour plot – and you realise that a similar figure is what you need to publish your paper in a high-ranking journal. You know the basics of flow cytometry, but admit you need a little…
Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…
The flow cytometer that we have all grown to know and love may have only come into its own in the 1990’s, but who would have known that the first cell sorter was invented as early as the 1950’s? With the recent death of one of the key developers of fluorescence activated cell sorting (FACS),…
For those of you who print out the results that you have acquired from the Flow Cytometer to stick in your lab book, did you know that inkjet printers and cytometers have a shared history? Flow Cytometry is less than 50 years old and machines today still use some of the same principles as the…
So, you’ve just received a call from the core facility that you hired to prepare and sequence your libraries. The facility director tells you that the sequence data from your next generation sequencing (NGS) experiment does not look good. You panic and, perhaps, let loose a scream of frustration—aaarrrrggghhhh! This project was going to be…
So you have some blood stored in the -20C or -80C and you want to isolate RNA from the samples. If you wanted DNA, you would have many products to choose from. But for RNA, your choices are more limited. Obtaining RNA From Frozen Blood is Difficult Why is that? The reason is that most RNA from…
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