How can I use the facility’s instruments?
New users of the facility should fill in an account creation request. All new users need to go through training before using the instrument.
How can I be trained to operate the instruments?
All users of the facility’s instruments must go through the training process regardless of prior experience. Training to operate the bench-top analyzers is a three-part process.
- Part 1 includes the 1.5 hour Flow Basics lecture, which is held the first Wednesday of the month in the conference room (reservation required by completing the new user form).
- Part 2 takes place at an appropriate instrument using samples you have prepared to give you hands-on experience; we require one session of hands-on training before granting access. If needed, we will offer one more session free of charge. After that, you can decide if you would like to operate the instruments yourself, or take advantage of our drop-off service.
- Part 3 of the training process tackles data analysis using FlowJo from the data you collected in part two, and Flow data base.
Complimentary training sessions (composed of 4 different modules) will be offered by the Flow Facility staff for self-operation of the FACSAria II SORP. Go to CRI PPMS and fill in an account creation request to begin the training process.
Which flow cytometer analyzers do you have?
We have the following flow cytometers in the Facility:
- BD FACSCanto II (3 lasers, 10 colors)
Which cell sorters do you have?
We have the following cell sorters:
- BD FACSAria II SORP (4-lasers) in a class II biosafety cabinet
- BD FACSAria II SORP (5-lasers) in a class II biosafety cabinet
How do I book a sort?
If you are a new user, fill in an account creation request or contact the facility. Go to the CRI PPMS website calendar, log in and reserve the time you need on the Aria calendars. Sorts can be only booked three weeks in advance.
How do I need to prepare my samples for sorting?
Please see Sample Preparation for Sorting.
How long will it take to sort all my cells on your sorter?
The answer to this question will vary greatly. You can make some rough estimates that should put you in the ballpark. For the typical low pressure sort with the 100µm nozzle you can estimate that we’ll run a threshold rate of about 3000 cells/second. This translates to about 10 million cells in one hour. For the typical high pressure sort with the 70µm nozzle you can estimate around 10,000 cells/second, translating to around 35 million cells in one hour. These estimates are conservative and speculative. We’ll make adjustments to the rate of cells/second based on the efficiency and quality of the sort in relation to each investigator’s needs and the situation.
Which pressure/nozzle settings are right for my sort?
This will depend on the size and characteristics of the cells more than anything else. The nozzle size should be about four to five times that of the cells that are being sorted. For sorts targeting most lymphocytes, you should run the 70µm nozzle. For larger cells (monocytes), you should probably choose the 100 µm nozzle. Increased pressure and the smaller orifice of the 70µm nozzle will expose your cells to a slightly harsher environment, albeit very briefly. But with 100 µm nozzle the threshold rate will decrease a lot compared to 70µm so the number of cell sort in an hour will be 1/3 less with 100µm nozzle compare to 70µm nozzle.
How do I delete the doublets?
Doublets can easily be eliminated by using a pulse geometry gate (such as FSC-H x FSC-A).
Where do I put my gates?
The controls for the experiment are critical for ensuring the proper cells are identified. A Fluorescent Minus One (FMO) control, for example, is critical for identifying the proper placement of a gate in a multi-color experiment. The spread of the data due to the fluorochromes in the panel cannot be corrected using an isotype control, for example.
How many events should I record?
The number of events is critical, so be sure to record the right number for your statistic analysis. Here is a table (Allan and Keeney, J Oncol 2010) showing how many events you need to record, depending of the frequency and the accuracy you want. To have an accurate number of cells, also use TrueCount beads.
How do I sort rare cell populations?
Watch the instrument to ensure success. Keep the core stream tight: the tighter the core stream (low differential pressure), the tighter the CV on resulting data, and the easier it is to determine the best placement of gates for sorting. Tight core streams also reduce coincident events (two cells passing through the intercept at the same time). Since flow cytometry requires single cells for proper analysis, the more doublets, the fewer sorted cells.
Eliminate the aborts. Aborts arise from a cell arriving at the laser intercept while the electronics are processing the previous pulse. This new event is lost (aborted). This results in a loss of cells as well as decreased purity. Optimizing the window extension for the cell type can have a dramatic effect in improving the quality of the sort and reducing the abort rate.
Avoid the artifacts. It cannot be stressed enough that a simple viability dye improves the quality of the sort for downstream applications by reducing the missorted false-positive cells that are dead.
Avoid the aggregates. In establishing a gating strategy, consider adding a pulse gate (height vs. area, for example) to eliminate cells that are aggregated.
Gate for success by using the appropriate controls for gate placement and avoiding the use of the histogram. Histograms mask data and make it impossible to find rare events.
Pay attention to the threshold level. When a threshold is set up, the cytometer is blinded to any signal that is below that threshold value. So, while it is possible to eliminate debris from the data using a high threshold, if the cell sorter cannot see the debris, it will find its way into the sort tube and contaminate the cells for downstream applications.
When setting up a gating strategy on a cell sorter – either with an operator or alone – make sure the steps above have been addressed. This will help ensure a good sort, even for rare populations.
How can I get FlowJo on my personal/lab computer?
The Flow Cytometry Facility administers the CRI site license for FlowJo analysis software. This results in a significant discount compared to individual purchase. To participate in this program you must be affiliated with UT Southwestern and be able to provide a valid account number for this license is to be billed. To obtain a serial number for your computer, download and complete this form and email it to firstname.lastname@example.org, or you can request it at CRI PPMS. Attention: You will be billed for a full year license if you request the software. Our billing cycle starts in September of each year. Using the software one time during the cycle constitutes a full year subscription.
How do I transfer my data on my personal/lab computer for analysis?
Use Cytobank to transfer your data. Cytobank is a safe way to manage, search, backup, share and analyze your FCS files over the web.
What services can Core Personnel Support Services provide?
- Experimental design.
- Protocol development.
- Instrument operation.
- Data analysis.
- Graphical production.
- SpaaS. The facility can provide full services for your study: experiment design, protocol development, samples staining, instrument operation and data analysis. This is particularly valuable for clinical flow when all steps must be standardized and equipment or personnel need certifications. Please contact the facility for more information.