How To Design Accurate & Effective Flow Antibody Panels (or, What’s An OMIP?)
I was at a meeting talking about the principles of panel design.
At the end of my talk, I had an investigator approach me and ask why he was not making progress on his 15-color panel that he started developing. So, I asked how long he’d been working on it.
That was his response. This might shock some of you but a month is not very long when it comes to designing an accurate and effective antibody panel for a flow cytometry experiment. Multicolor panel design requires a delicate balance of biology and physics. Understanding the biology of the system and the physics of flow cytometry are critical to success.
Antibody panel design (and flow cytometry experimental design in general) is a complicated process that can take a very long time. There are, however, some things you can do to simplify the process and shave weeks if not months off of your design time, including:
1. Knowing your biological question.
The driver in this whole process is knowing what the question is. This question will help determine... Read More
10 FlowJo Version X Hacks That Will Help You Publish Your Flow Cytometry Data
So you just got the most amazing results of your life and you can wait to show it off in lab meeting, or create the figure for a publication. Here are a couple of tips that will help you ensure that everyone else also sees how amazing your data is!
1. If you only have a few events, use the option to “show large dots”.
When you only have 4-5 events in a population, it can often be difficult to see. If you turn off the high resolution, you can see the data better.
Double click on a plot in the layout editor and under the specify tab, check the box for ‘Use Large Dots”
2. Make sure your axes are labeled properly and cleanly with something that makes sense.
Many cytomters have a default axis label like FL1, or 525/50, as two examples. Some scientists doing flow are savvy enough to use the $PnS keywords to enter in their own axis labels during acquisition.... Read More
Top 10 Flow Cytometry Resources
I’ve been in the world of flow cytometry and cell sorting for a very long time. Now, don’t worry, this isn’t going to be some lament about the “good old days.” Well, maybe just a little. But there will be helpful takeaways, I promise.
I was trained by the incredible staff in the core facility at the University of Arizona. When I moved to UC Davis and proceeded to start up a core facility, there were very few resources to guide me. No Google communities and LinkedIn groups. Email was new, there was the Purdue Cytometry List, but that was about it.
You certainly couldn’t get a degree in flow, and other than the annual course that alternates between Bowdoin and now Albuquerque, there were very few courses in flow. Even the publications were few. For example, I made the following simple graph of publications mentioning flow cytometry by year:
4 Fluidics Tips That Will Change Your Flow Data For The Better
Friday is the 4th of July in the US – and we celebrate that day with picnics, spending time OUTSIDE the lab and fireworks. And our Independence, but I’m not up for a political discussion right now. For flow geeks, fireworks are like flow cytometry – they happen in the dark, they are full of many bright colors, and we’re all looking for the patterns the colors make. So in honor of a day outside the lab, it seemed appropriate to talk about fluidics… going with the flow for best results.
A flow cytometer has three major components – fluidics, electronics and optics. From setting the run speed (‘flow rate’), to cleaning the instrument after a run, to changing the sheath fluids, the typical researcher interacts mainly with the fluidics side of the system.
The majority of flow cytometers on the market use a differential pressure to move the fluids and cells around the system. In these systems, the pressure of the sheath fluid sets the speed of the flow. The low, medium and high buttons on the instrument change the differential pressure between... Read More
Counting Cells Will Save Your Flow Cytometry Experiment, If You Do It Like This
Fast, accurate or efficient – pick two. How to decide when you can’t have it all.
The hemocytometer is considered the gold standard for cell counting. Invented by Louis Charles Malassez, this precision etched microscope slide can allow the researcher to count their cells under the microscope with amazing accuracy. It is inexpensive, relative to other methods, but is by no means the most efficient or fast method out there.
The single biggest key to using a hemocytometer is training, training and more training. Since the investigator is visually inspecting the cells within a boundary, the rules of what cells to count and what to exclude on those boundaries becomes critical.
If counting more than one sample, proper cleaning of the hemocytometer is a second critical step. If the cleaning solution is not removed completely, it can cause cell lysis... Read More