Written by Tim Bushnell, Ph.D
Flow cytometers have three major components:
- Fluidics that move the cells from the sample tube to the intercept point
- Optics that collect the light
- Electronics that convert the photocurrent into a digital value that is stored for later analysis
If we look at the fluidics system of a standard flow cytometer, laminar flow is established by the sheath fluid. This is even flow with fluid flowing in ‘parallel layers’. Due to some friction on the side of the tubing, the flow ultimately develops a parabolic shape, as shown below.
Into the center of this laminar flow, the cells are injected. The cell and fluid mixture is introduced at a higher differential pressure, keeping the cells in the center of the laminar flow, and allowing the process of hydrodynamic focusing to occur, which causes the cells to spread out along the velocity axis, single file, as they approach the intercept point.
After the intercept, the cells either flow into the waste or, in the case of a cell sorter, the stream is broken into droplets, and the appropriate droplet is charged and sorted.
Most of the interactions that a user has with a flow cytometer is with the fluidics system, and many of the issues that users will face in troubleshooting problems on the instrument will also be here.
Here are four important questions to ask yourself when trying to understand and troubleshoot the fluidics system in your flow cytometer…
1. What sheath fluid are you using in your cytometry protocol?
Many institutes run a phosphate buffered saline (PBS) as their sheath fluid. This can be made in-house or purchased from any number of vendors. However, since the sheath fluid and sample stream do not mix, it’s not necessary to use PBS for the sheath fluid.
Others use just water for the sheath. Several years ago, we converted to using water with 0.1% 2-phenoxyethanol, which is used as a preservative and has some surfactant properties.
For cell sorters, however, one must use some buffered saline solution for the sheath fluid. Since 2004, I have used a 10 mM HEPES buffered saline solution for my sorting needs. This is because HEPES is a better buffer at sorting pressures than Phosphate.
If your cells are prepared and held in a culture media (like RPMI), adding HEPES to the solution is appropriate, as culture medias are typically formulated to buffer in a CO2 environment.
Our solution (10X) is as follows:
- 13.015g HEPES sodium salt
- 11.915g HEPES free acid
- 80g NaCl
- pH to 7.2-7.4, final volume to 1L
2. What is the differential pressure?
The core stream is where the action occurs. The cells, contained in the core stream, are spread out along the flow axis until the cells become single file as they pass by the laser intercept.
Since the sheath flow rate sets the speed of the system, the only way to increase the number of events seen by the flow cytometer is to increase the differential pressure between the sample and the sheath fluid.
The consequences of increasing the differential pressure include:
- Increasing the number of coincident events
- Increasing the spread of the data
In the data below, cells were run at three differential pressures, from low to high. As you can see, increasing the differential pressure increases the number of events (intensity increasing from left to right), but the spread of the data also increases.
Best practice is to consider running a low differential pressure at a higher concentration.
3. Does the data show backpressure or a clog?
On multi-laser systems, knowing the order of the lasers is a good thing. One impact of things that affect the sheath flow rate (i.e. clogs and back-pressure) is that the time to travel between lasers is impacted. Thus, as the signals are matched by the delay electronics, the resulting data will be wonky.
As shown here, this was the result of a problem with the sheath flow on a 4-laser instrument, and the green laser was the 4th laser in order.
Notice how the signal on the ‘poor flow’ plot bounces around. This indicates a major issue with fluidics and if this is observed, it is time to stop and do a quick cleaning/check of the system.
Remember, filtering samples is always a good idea!
4. Is the cytometer fluid pathway clean?
Cleaning the flow cytometry fluidics pathway is a thankless task. Each vendor has their own recommendations as to how often to clean the system, and what reagents are best to use.
A long clean of the system should occur at least once a week (more if it is a heavily used system), and a shorter cleaning should occur daily before use. This is in addition to any cleaning that is done between users.
In long cleaning, one should bypass any in-line filters, so that the cleaning solutions do not compromise the filter status. This process takes about 1.5 hours and uses a detergent (Contrad), an alcohol (Ethanol), and sheath fluid.
- 1% Contrad 70 for 15 minutes (in sheath tank and Sample Injection Port)
- 70% Ethanol for 15 minutes (in sheath tank and Sample Injection Port)
- Water for 30 minutes (in sheath tank and Sample Injection Port)
- Sheath fluid for 10 minutes (in sheath tank and Sample Injection Port)
- Run QC particles
One important thing to remember is that when bleach is used, it is critical to wash out the bleach before opening the machine for general use.
In this experiment, 10% bleach was run for 5 minutes on the SIP before removing the tube and placing a tube with water on the SIP.
The system was either run for 5 minutes (blue line) or not run at all (red line). Peak 6 beads were run and 10,000 single events recorded. The data shows that in the presence of residual bleach, the APC signal decreases by 50%, while the PE signal is relatively robust (only a 2.5% decrease).
A quick fix for this is to put a fresh tube of water on the SIP and start running the system while you set up the electronics — and this issue will be avoided.
As a side note, there are some other fluidics arrangements out there — such as the Attune, which uses an acoustic wave to focus the cells in the center of the core stream, and the Guava instruments, which use a microfluidics capillary system, meaning no separate sheath fluid.
These types of issues can also arise on these systems, so watch your data.
Understanding the fluidics system and observing the consequences of the system during acquisition is important to solve problems before they become major issues. While walk-away options exist on cytometers (such as high throughput sampling systems), care must be taken to make sure that the samples are properly prepared so that clogs and other preventable nuisances are avoided and data is not lost.
To learn more about how to troubleshoot the flow cytometer fluidics system and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.
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.