If you are working with a shared flow core facility, then you probably know how important it is to get the most out of your limited cytometer time. Aside from costing your lab time and grant money, shared cytometers are a valuable resource so booking additional time may be difficult. Therefore, we have compiled this list of tips for maximizing your productivity during your time at the flow core.
Sections:
Experiment Design
Before you consider booking time at your core facility, you first need to design an experiment with the following factors in mind:
Cytometer, Laser & Filter Selection
There is no point wasting time designing experiments for equipment that you do not have. So start by understanding what cytometers are available for you to use. If you’re lucky enough to work with a core that offers a variety of cytometer options, then you may need to consider which one is the “best” for your planned experiment. FluoroFinder’s new cytometer page lists many analyzers and sorters with basic info on their capabilities and performance. Also, your core facility website will likely have detailed information about the setup for each cytometer. Consider how many parameters you plan to investigate and select an analyzer or sorter with the appropriate laser/filter configuration, or consult your core manager for their recommendation.
Marker, Color & Reagent Selection
One of the most common complaints from core managers is researchers wasting time designing experiments that won’t work on their core’s equipment. FluoroFinder eliminates this issue by pre-loading your core’s laser and filter configurations into the app. Therefore, researchers who design multicolor flow experiments on FluoroFinder can be sure that their selected colors and reagents will work in their core. Video: Designing a Panel on your Cytometer
Controls, Viability Dyes & Dump Channels
Selecting the proper controls is also a simple but important step. Consider using:
- Instrument controls to adjust instrument voltage gains and compensation.
- Gating controls to distinguish between specific and non-specific binding.
- Experimental controls as necessary.
Are you analyzing a live cell population or looking at a specific organelle? Consider using viability dyes or organelle-specific stains for your cell population. Video: Using Viability Dyes in FluoroFinder?
PMT Voltage Settings
PMT voltage should be set to maximize signal-to-background resolution. Setting the voltage too low may result in sub-optimal signal detection, whereas setting it too high may result in the positive population appearing off-scale. While you may use beads or analysis software to determine the optimal PMT voltage settings for your cytometer, we recommend consulting your core manager for their recommendations before running your sample.
Flow Cytometry Software
There are a number of different software options for running and analyzing flow cytometry. If you are ever unsure about how to use software, we recommend consulting your core staff rather than wasting valuable core time learning a new user interface.
Core Etiquette
Always remember to respect the equipment, the core staff, and your fellow researchers. Like any shared resource, the core facility runs best when users follow a simple set of rules.
Booking Time
When booking your core time, be sure to factor in your setup and cleaning time. Don’t expect to walk into the core and begin running your samples immediately. Be sure to consult with your core manager regarding the appropriate amount of time to assign for these important tasks.
Sample Preparation
Flow cytometry experiments usually require a significant amount of sample preparation. Any necessary cell suspension, filtration, etc. should be performed prior to heading down to the core facility to run your sample. Also, be sure that your collected population is sufficient for your planned analysis. If you are working with live cells, consider how you will keep them happy (temp, pH, etc.) in the core as you prepare the cytometer for your experiment. For more details, please see our article on pre-sort sample preparation.
General Recommendations
Remember that the core facility is a shared space so you may need to be even more mindful than in your normal lab. Obviously follow good lab practices and never bring food or drink into the facility. Remember to clean up any buffer spills, etc. as necessary.
