Standard Operating Procedures
Before you ask questions or make a request, please familiarize yourself with our guidelines on:
- Training on a Machine
- Booking a Machine
- Machine-specific Protocols
- Biological Sample Considerations
- Generation of Data and Backups
To schedule resources for this core, please login to iLab.
Training on a Machine
The level of access you are permitted to the equipment depends on training and user experience. Without exception, all users wishing to operate the FACS Fortessa or LSRII must be approved by the core director; requests for training go through the director or staff. You must obtain proper training before conducting any experiments using flow cytometry.
The Analyzer Class applies to the Fortessa and LSRII instruments. It provides a level of training that makes users able to function on the instruments with occasional assistance from the core director or staff. Completion of the Analyzer Class by itself does not mean an individual is certified to run the instruments without supervision. To become an unassisted user, you must demonstrate an independent ability, this will be determined by the director or staff.
Sorters (such as the Ariall) are far more intricate and complex than the analyzers and training individuals with existing heavy and active sorting needs is only undertaken on a case-by-case basis. Please consult the director.
These two steps are necessary for the successful understanding and operation of the instruments.
- We recommend that all users review our educational materials on the principles of flow cytometry, as well as other online resources. Please download and read the Introduction to Flow Cytometry: A Learning Guide.
- In addition, self-paced tutorials on the FACS Diva software are also available from the Facility Manager.
Once these two steps are completed, individual instruction is available with the facility manager. If users in your group have need of the machine more than once a week, it would be best to have someone in your lab trained to use either the FACS Fortessa or LSRII.
The user must be fully informed of start-up procedures, cleaning and shut-down procedures for the specific instrument they are using. Failure to properly clean the instrument may result in revocation of user privileges. Users are encouraged to discuss experimental design with the facility manager, particularly if multiple fluorochromes are employed.
We encourage investigators to run their own samples on the FACS Fortessa and FACS LSRII because this gives the investigator a better understanding and more control of their experiments, data and the limitations of the technology.
Lastly, training is not always needed. If an investigator has 3 or 4 experiments they need to do, it would not be worth while to take training. It would be easier to have the core staff assist in completing those 3 or 4 experiments. However, if flow cytometry is to be part of one's research program, then it would be advantageous for the individual to have training and become independent on the analyzers.
Booking a Machine can only be done in iLab.
All four machines must be booked in advance. Booking is only done through iLab. You must first have an iLab account which can only be done through your divisional financial manager. Once the iLab account is established, you must apply for access to the core(s) to which you will need access. Once you have access to the core, it is then suggested if needed, you sign up for training.
If you are on the assisted list, where core staff is needed, you are limited to the working hours of 9 am to 5 pm. Unassisted users can use the machines at their leisure.
There is a 14 hour block on sorting requests. Same day sort requests are discouraged and if truly needed must be done in person. Understand that with the schedule of core staff, there is a 50-50% a same day sort request will be denied.
When you book a cytometer (and operator time), the type of sample and types of fluorochromes must be stated. Please also indicate in the description of your experiment whether facility manager assistance is needed or required. Always include your contact information in your booking.
It is expected that you will conduct your experiment such that you can show up on time and finish on time.
If in doubt, the core would rather you overbook time than under book. Under booking time will create problems with other users.
IF you are more than 50% late of your booked time, you run the risk of losing your booking entirely and having to move to the next available slot that can accommodate the number of samples you have.
As a rule of thumb - plan 1.5-2.5 minutes PER sample. Multiply that by how many samples you have, and add an additional 10 minutes of set up time for the experiment. This is a starting framework as to how much time you should book for your experiment.
Booking the FACS Fortessa/LSRII
Limitations and etiquette guidelines for this machine are simple.
- Instruments may not be booked for more than three-hour blocks. Booking consecutive blocks (6 hours) may only be done with the Director's prior approval. This is to prevent monopolization of the instruments. Users are expected to be considerate of the other users.
- Users must book the time the procedure is expected to take. If the estimated time grossly underestimates the actual experimental time, the Director reserves the right to truncate the experiment if someone else has signed up for the next block. The user that is running overtime must consult with the user signed up for the next block. In addition, booking more time than is needed is an equally unacceptable practice.
- If a user is more than 30 minutes late and does not inform the operator or flow center, the user risks losing their booking time at the discretion of the Director.
- Kinetic or time course experiments are allowed, but users must be considerate of other users signed up for the machine. In addition to booking time, please confer with the Director when conducting a kinetic or time course assay. Assays expected to take longer than three hours should be booked in the late afternoon to be considerate of other users.
- Core Staff reserves the right to adjust and/or move an appointment at their discretion.
Booking the FACS Aria/Sony MA900
When booking time for the sorter, both operator and user have an implied contract to be ready at a certain specified time. Adjustments due to procedural issues or instrument issues may be cause for delay on either side. Communication is critical to keeping the appointment; however, certain situations may alter the arrangement between the user and operator.
- If the user is more than 30 minutes late and does not inform the operator, the user risks losing their booking time at the discretion of the operator. If the user is from outside or it is otherwise a fee-for-service use, in the case of a no-show the user will be assessed the set-up fee (1 hour) for sorting whether or not the sort takes place.
- Experimenter must fully inform CoreStaff, preferably via the iLab sign up sheet, all details about the sort including cell type, type of sort needed, ALL colors used and how many samples are to be sorted.
- The user must book the actual time the procedure is expected to take. If the actual experimental time grossly over- or underestimates the required time, the Facility Manager reserves the right to adjust the booking and/or truncate the experiment.
- In booking the sorter in ilab, not only must the type of sample and types of fluorochromes be stated, but the type of sort must also be included (for example, bulk sort versus a sort into plates). Experimenter is expected to bring all needed controls for an experiment including unstained, single color compensation controls, and needed FMO (Fluoresce minus one) controls.
- Core Staff reserves the right to adjust and/or move an appointment at their discretion.
These protocols cover user-operable instruments, specifically the FACS Fortessa and LSRII. The Director and staff are responsible for maintaining quality control and monthly cleaning of all the machines. This involves swapping the sheath tank for various cleaning solutions, which takes as much as three hours and removes the machine from service. Still, it is the responsibility of all users to follow the protocol outlined in cleaning the LSRII/Calibur after each experiment, even if another user is booked on the machine the same day.
Fortessa/LSRII (Start-up Procedure)
- Check sheath level, make sure there is enough for the duration of your experiment.
- If sheath tank needs to be filled, empty waste tank.
- Turn on the instrument.
- Turn on the computer and set up data-acquisition protocols.
- Prime instrument once.
- Let instrument warm up 15 minutes prior to use.
Fortessa/LSRII (Shut-down Procedure)
- Run a tube with 2 mL of 10 percent bleach.
- Move swing arm to the side to aspirate 1 mL of bleach.
- Move swing arm back to center position, put the instrument on RUN and on HIGH, and leave it for 5 minutes.
- Put a tube with 2 mL of distilled water on the instrument.
- Move swing arm to the side and aspirate 1 mL of water.
- Move swing arm back to center position, put the instrument on STANDBY, and leave it for 5 minutes (laser cool-down).
- After 5 minutes the instrument may be turned off (or left for the next user).
IMPORTANT: Do not leave more than 1 mL of water in the tube when you turn off the machine.
Biological Sample Considerations
Users of the cytometry facilities must not run any sample over a jet-in-air sorter containing a potential biohazard without first informing the Director and staff. Requests to run a sample containing a known or potential biohazard must be given, in writing, to the Director for review by the institutional Laboratory Operations Safety Officer and Flow Core Director.
The written statement must answer the following questions.
- What is the aim of the project?
- What is the nature of the cells in question (species, tissue source and so on)?
- How have the cells been treated or processed by the user?
- For cells of human origin, has the sample been screened for HIV and HepB (both mandatory) or for other infectious agents?
- What is the nature of all biohazards associated with the sample (inhalation hazard for example)?
If the sample will be run on the FACSFortessa or LSRII cytometer, appropriate containment procedures must be followed regarding the waste. It is imperative that the user follow the cleaning procedures so the analyzers are decontaminated afterwards.
Under no circumstances are radio-labeled samples allowed within the Flow Core Laboratory. Anyone running samples that are found to be radio-labeled will be permanently banned from the facility.
For single cell preparation, consider trypsin and see if trypsin cleaves the molecule in question. If not, trypsin can solve a lot of problems. However, if trypsin does cleave a desired surface protein, consider: 1) plating the cells on collagen and using collagenase to harvest; 2) Accutase from Phoenix Flow systems; 3) TriplE; or 4) Versenne solution (variations of 1-5mM EDTA in PBS). All combinations of the above must be tested for the best times and incubation temperatures that have the least affect on cells. Consult core staff.
Always consider the use of a viability dye in your experiment. Dead cells bind antibody and thus will "light up" in a flow cytometer. Consult core staff, but many reagents are available for dead cell exclusion (ex., DAPI, SyTox Blue, for example).
A sorter can never be made sterile. There is no such thing as a sterile sort. We do take efforts to make sure the machine is as clean as possible and is cleaned between users. This involves a combination of 10% bleach, as well as 70% Ethanol. Sample line is rinsed between clients with 70% EtOH. Machines are periodically taken off line and run entirely with 70% EtOH as "sheath".
It is strongly recommended that one doubles (2X) the amount of antibiotics if culturing cells post sort. It is rumored that the half life of Penn/Strep is about 18 hours. One would be advised to change the media if possible. Also, consider using Gentamycin in lieu of Penn/Strep. It has a longer half life and is thought to have anti-mycotic properties.
The core absolves itself of all responsibility for contamination if the user insists in not using any antibiotics at all.
Generation of Data and Backups
Large amounts of data are generated and stored on the FACS Fortessa, LSRII and Aria's.
Each user and user group is responsible for the backup of their own data from these instruments. Additionally, users and user groups are responsible for the media used for backing up the data. The flow center does not provide flash drives, CD-Rs, or DVD+Rs.
All Flow Cytometry computers have a second storage drive (drive D:) for archived data. All data from the second drive that is older than one month may be permanently removed on the last business day of each month. Core staff will back up data from the Diva Browsers periodically.
ONLY Flow Core Staff is authorized to remove data from the Diva Browsers. Core provides a "courtesy" backup.