There are some wonderful toys in the lab that enable us to open up a whole new world in science. One of those is a rather pricey and an incredibly sensitive laser-based apparatus capable of counting and sorting cells, detecting biomarkers, and engineering proteins: the flow cytometer. By propelling cells through the path of the laser in a stream of liquid at high speeds, the electronic detection apparatus is capable of rapidly analyzing, counting, and separating cells at a speed and precision that scientists across many fields now rely on and utilize on a regular basis.
Tragically, these instruments, because of their incredible speed and precision, require love and care that is often not given to them! With mishandling, these instruments can become badly clogged, rust, bent, worn, and eventually require expensive repair. This can be avoided for the most part, however, so let’s learn how! The machine is also often misused, less to the detriment of the apparatus, and more to the detriment of the user’s experiment!
Pin these rules on your machine. One sheet of paper could save you thousands of dollars and hundreds of lost hours as well as the humiliation of BAD science.
Don’t use concentrations of contrad higher than 10% as it may eat away the sealing of the flow cell.
Don’t let contrad “sit” too long in the machine for the same reason. After using contrad, ALWAYS rinse with distilled deionized (dd) water.
Don’t use a more than 10% concentration of sodium hypochlorite solution (household bleach) to remove cell debris. This is especially useful after a clump of cells has created a blockage.
Don’t use a solution with too high a concentration of cells. It’ll just clump and block the machine.
Don’t forget to do an extra rigorous cleaning after running yeast and bacteria.
Don’t skip on the monthly intense cleaning that manufacturers advise!
Don’t continually top up the cleaning fluids. Dump the last bit of the solution (see next point) and make sure the cleaning solutions, including the (dd) water, are changed daily.
Bubbles! Don’t let samples, controls, cleaning fluids, or anything else in your tubes run dry! This causes bubbles in the machine and can really throw your results off. They can be a pain to remove too.
Don’t overlook cleaning both the main microfluidic system and the secondary one. If you don’t know how to do both, ask a technician.
Don’t reuse the results of controls. You need to run controls every time. So many factors change day-to-day, including the temperature and air pressure, and affect how the machine will sort the cells,. A meaningful negative control is ALWAYS necessary for background determination. Single positive controls for each fluorochrome are REQUIRED in every experiment for EVERY TIME to calculate compensation. Reusing controls is pointless. So, if you’re going to do this, have a pajama day instead as it will do more for your science!
Don’t skimp on the time when cleaning. Clean things in 3–5 minute cycles. It takes time to remove debris and properly flush the machine of cleaning chemicals. If you’re running late, don’t skip the cleaning steps to hurry along for the next person. You may end up wasting a lot of their time as they troubleshoot or even ruin their experiment by wasting precious samples. It’s better to take 5–10 minutes at the beginning of their slot than waste their entire slot! You can also just tell them you didn’t have time to do the cleaning. Better to be nice and give them a heads up.
Don’t be caught with naught. If you expect to see to least 10,000 events, you’re going to need to start with a MINIMUM of 100,000 cells, but even this number is risky. It’s better to use extra than be stuck.
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 […]
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