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 the sample and sheath. 1. Faster IS NOT better. As the differential pressure increases (increasing from low -> med -> high) the size of the core stream is increased. This allows for more cells to pass by the intercept per second. However, that is not without consequences.
A. The spread of the data will increase due to the position of the cells within the laser interrogation point.
B. The number of coincident events will increase as more cells pass the intercept while the previous cell is being processed.
2. Plot time as a measure of quality. During acquisition, monitor how the sample is running by plotting time versus the fluorochromes being used. It is best to have a plot for each laser, so that if pressure related issues arise (which can affect the time delay between different lasers) they can be readily detected. 3. Sheath and the sample fluid do not mix … mostly. Because of the laminar flow that is established and how hydrodynamic focusing constrains the core stream, the sample fluid and sheath fluid generally do not mix. This means that solutions like water can be used for the sheath fluid. There is one place, however, where mixing can occur. That is at the point where the sample is injected into the running sheath fluid, before the hydrodynamic focusing takes place. Generally this is not an issue, but if performing a Calcium flux experiment, it’s important to ensure that this area of mixing doesn’t disturb the calcium equilibrium that is being measured. 4. Wash the sample injection port before starting. Many cleaning protocols use bleach and water to clean the SIP after each user. Sometimes (often!) users are in a rush and the water rinse is not completed. If this happens, the next user can lose valuable data as the bleach will negatively affect the fluorescence on the cells – especially those with APC. To prevent this, put a fresh tube of water on the instrument and run on high while getting the instrument setup for the next run. For those in the US, I hope you have a great holiday and enjoy looking for those patterns in the colored lights in the sky. For the rest of you, we’ll be back next week with more tips. Don’t forget to check our calendar of upcoming events.
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My other passions include grilling, wine tasting, and real food. To be honest, my biggest passion is flow cytometry, which is something that Carol and I share. My personal mission is to make flow cytometry education accessible, relevant, and fun. I’ve had a long history in the field starting all the way back in graduate school.
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