Standard Operating
Procedures

We work to provide the best possible flow cytometry outcomes for users of our services. To meet this goal, we put certain guidelines and operating procedures in place to make it simpler for both the users and our facility technicians to interact and achieve the best possible results.

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. 

  1. 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
  2. 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. 

Booking a Machine 
All four machines must be booked in advance. Booking is done via a MS Outlook Calendar. Booking from the Houston Methodist Research Institute webmail is not available.

If you plan to use the instruments on a regular basis, you should add the schedule calendar of the appropriate machine to your shared calendars. To do this, first contact the Director and request access to the Machine Specific Calendars. A PDF file will be sent to you. Click Open a Shared Calendar from within Outlook and type the name of the instrument you wish to use.

  • TMHRI FACS LSRII Calendar
  • TMHRI FACS Fortessa Calendar
  • TMHRI FACS AriaII Calendar

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. 

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.
  • Complete contact information must also be provided in case your booking needs to be moved or adjusted by the Director.

Booking the FACS Aria

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.
  • 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, 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).
  • Complete contact information must also be provided in case the booking needs to be moved or adjusted by the facility manager. 

 All users must have the following information in their bookings whether it is for analysis or sorting: 

  • Name
  • Extension number
  • Time of experiment
  • Description of experiment
    • Type of sample
    • Type of fluorochrome
    • Type of sort
    • Whether it is a biohazard

If you are only doing analysis, you do not need to worry about sorting conditions. The following figure is a quick reminder of the contact information you need to provide for bookings.



Machine-specific Protocols
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. 

  1. What is the aim of the project?
  2. What is the nature of the cells in question (species, tissue source and so on)?
  3. How have the cells been treated or processed by the user?
  4. For cells of human origin, has the sample been screened for HIV and HepB (both mandatory) or for other infectious agents?
  5. 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.

Generation of Data and Backups
Large amounts of data are generated and stored on the FACS Fortessa, LSRII and AriaII. 

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.