Also, Please refer to my previous article, for steps to implement to ensure that a sample is ready for flow cell sorting. Here, I discuss some of the checks and measures that you can do post-sort and how to effectively interact with your core facility personnel.
It is human to err but you might find it difficult to use that as an excuse in your next lab meeting. Here are a few checks that you can do to ensure that all’s well that ends well with your flow cytometry experiment.
Re-Evaluate Your Catch Tubes
Sorting is strenuous not just for you, but also for your cells. Post the laser interrogation point, a lot of factors, such as nozzle size and pressure, temperature of the catch buffer or plain bad luck, can damage your cells before reaching the catch tubes. Therefore, re-run the catch tube as a sample to ensure that the cells fall in the corresponding sort gate. However, this might not always happen since you already excited the tagged fluorophore and it might not possess the same peak emission by the time you analyze your catch tube. Ensuring slightly relaxed sort gates will go a long way in helping you heave a sigh of relief.
Fragile! Handle With Care
Cells that have just come out of the nozzle are as vulnerable as a dodo at the edge of a cliff. Don’t take them for a spin just yet. Allow them some time to settle down. Cell sorters are usually housed in air-conditioned rooms wherein the room temperature hovers between 19-22°C. Give your cells time to come to a cozy 37°C before toying around with them.
Statistics to the Rescue
Strange as it may seem, the best yardstick for a sorter’s performance is its post-sort recovery than purity. Andrew Riddell and colleagues have come up with a unique parameter called Rmax that can help calculate the sort recovery based on several parameters. They have also designed calculators which help you calculate the Rmax value for your particular sort. Their paper is an interesting read. Facility managers might benefit more from this but it pays to know about it as an end user.
You Can Still Enrich
Sometimes, despite the best optimizations, you may end with far less purity than desired. One way to get around this, is to put the cells back in culture and allow them to grow to a sizeable population before re-attempting the sort. If you already know that you have insufficient counts of cells, grow them in culture till they are in suitable numbers and try to enrich for the target population with a magnetic sorter. Then, continue with another sort to get better purity and/or yield.
Interacting With Facility Personnel
No matter the ingenuity of your science or the capabilities of your core facility, your results are only as good as your peer-to-peer relationship with the facility personnel. A flow cytometry facility usually lays out strict guidelines in the interest of accessibility, smooth operation, and high uptime of the facility. Cytometrists are some of the coolest nerds on the planet (I know, I am one) but they can do little if the user doesn’t do his/her homework properly.
Discuss to the Max
It is important to discuss all the facts pertaining to the experiment well beforehand with the facility manager or operator to ensure smooth sailing. Note down any concerns of the operator and be sure to let him/her know about your limitations as well. Remember that the operator might perceive a less than one-log decade shift in fluorescence intensity as inconsequential. However, it could mean a life’s worth of work for you. Remember, it doesn’t hurt to put your PI in the loop as well.
Log Your Usage
Ensuring that instrument usage is properly logged and documented is a collective prerogative of both the end user as well as the facility manager. Proper logging ensures traceability and proves extremely useful in the event of a disagreement. Usage logs also serve as case studies and help the service or application support specialist in troubleshooting any issues faster. Any changes midway during the sort or away from the norm should be invariably mentioned in the log book.
Assertion Is Key
This goes both ways. Remember, that as an end user, you have every right to ask for calibration and QC reports prior to running your samples. Have a look at these reports and ask the operator to explain to you the implications of any variations, if any (and while you are at it, pass on the information to your PI as well). As facility personnel, you always have the right to raise a flag if the user requests more than what is in the original written plan. Same thing goes when you suspect the sample doesn’t play well with the instrument. Again, if changes are made midway upon request, you need to ensure that the user documents this in the log book and signs it. Never let users simply take a facility’s time for granted.
Unfortunately, many core facilities suffer from the bane of workplace politics, nepotism and fund crunches. While there is little that you can do to change the situation, ensure that it doesn’t get in the way of your work. I have seen labs that don’t play too well with core facility personnel or claim entitlement rights over instruments. Although rare, these things have the potential to hamper scientific work. Ensure that you strive to maintain a good working relationship. If in disagreement, it is better to put it in writing, with appropriate people in the loop, than verbally complicating it.
Cell sorting is both art and science and takes years of learning to perfect. It is even more challenging when you encounter new (or strange) cell types which might require you to unlearn everything and start from scratch. Therefore, keeping abreast with the latest in this field is of paramount importance to stay ahead of the curve.
Getting efficient sorts requires a lot of skill – both on part of the operator as well as the end user. What I have described are just some of my observations as a facility manager having had the experience of manning a great core facility.