Golden Rules For Cell Sorting


The intended audience for most of these “rules” is the universal flow cytometry core facility staff member. They were developed for training and educational purposes over the last 10 years. However, in order for everyone’s expectations to be met within the University of Utah core, I believe this list is applicable for both core facility staff as well as the client.

Be absolutely ready at scheduled appointment time, not 5 minutes late, not 2 minutes late
-Clients will notice that it’s not that important to be on time if you’re not ready on time
Preparation:
-Instrument calibrated, cleaned, rinsed
-Experiment opened and ready
-Correct parameters selected
-Relevant display plots opened

It’s all about the sample!!! And it’s all about yield
-Save the bare minimum number of events to allow you to draw appropriate gates for sorting.
-Once gates are set, save more events if needed while sorting
-If you’re running a few samples that will be using the same gates (ex. GFP), start sorting the second you realize that the sample looks good, then save the file.

For complex sorts, never assume you’re done once you started sorting.
-While sorting, spend 10 minutes looking through alternate display plots making sure you have the best possible gating scheme.
Ask yourself:
-Did minor compensation issues cause cells of interest to be partially lost through the gating scheme?
-Is your light scatter gate too small or too large? i.e. missing activated cells or incorporating aggregates?
-Are your live cells slowly creeping up in the dead-cell channel? (or something like that – where live cells slowly get brighter in the PI channel even though they’re still alive)

Being the client’s friend and striking good conversation is a distant second to completing the sort with the highest possible quality
Problems that result from not paying attention:
-Running tubes dry leads to bubble introduction into flow cell, which leads to bad purity and delays
-Forgetting to save a data file
-Forgetting to start sorting
-Time delay results in poor viability and delayed scheduled appointments downstream
-Inappropriate conversation causes people to avoid your lab
-Let’s face it, kids these days can’t handle thoughts longer than twitter length
anyway 🙂

Work with the client to fully appreciate the biology of the experiment!
-A firm understanding of the instrumentation is a small component of what you can contribute to a project

Sometimes the client is right, sometimes you are.
-Work together and be humble. There is always the chance you are wrong
-Often times the investigator is sorting based on another publication with incomplete information.
Example A. Client is trying to reproduce publication stating an antigen to be negative on a cell when its actually dim positive, but less bright than it is on another cell population. This is crucial biological information that can impact how gates and plots are set up.
Example B. The nomenclature and immunophenotype of many common cells are far from standardized. Think Monocyte subsets and Dendritic cells. What you may be familiar with as a specific immunophenotype may not be accepted by another group.

Maximizing available instrument time.
-If you know you’re going to get done with the current sort early, call the next person to see if they can come in early.

I see no need to ever draw a sorting gate based on a histogram

Rarely is a rectangular gate the ideal choice.
-When is the last time you saw a rectangular population?

Filter sample right before you sort, not an hour before back in your lab.

Use appropriate FMO control for dim antigen gate setting

Never sort cells into an empty tube

Never be 100% sure
-We have a tendency to apply something that we ‘know’ from one model system to another, to our own downfall.

Work to foster a warm collaborative environment within the lab

Always have client confirm gates

Follow-up with client about previous sorts
-Make sure they have access to the raw data files, as well as a printout (PDF) of the actual sort gates and post sort purity check.
-If available, a printout (PDF) of the sort report is helpful.  How many cells were processed?  How many cells were sorted?  Calculated efficiency? Final Purity? Final Viability? Final Cell Count?

Develop a simultaneous purity check and cell count protocol
-Over and over you will have to answer the question of “where did all the cells go?” If you do a purity check with Accucount beads, you will know exactly how many cells they walked out of your lab with.

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