Houston Methodist. Leading Medicine.
Houston Methodist. Leading Medicine

Standard Operating Procedures

Flow Cytometry Laboratory Operating Procedures

The Flow Cytometry Laboratory strives to provide the best possible flow cytometry service to its users. In order to provide this service, certain guidelines, or operating procedures must be in place for the benefit and understanding of both the users and the flow cytometry facility.  The key to having these operating procedures working is communication and dialogue between the Flow Cytometry Laboratory and the user.


Training

The level of access to the equipment is dependent upon training and user experience. All users wishing to operate the FACS Fortessa and/or LSRII must be approved by the Core Director. There are no exceptions to this rule. Being shown how to use the machine by a lab mate does not qualify one to operate these instruments. If one is a novice, you must obtain proper training before conducting any experiments using flow cytometry. All requests for training for these instruments must go through the Core Director (David Haviland, dlhaviland@houstonmethodist.org) and/or his staff.

The Analyzer Class only applies to the Fortessa and LSRII instruments. It provides training to get users operational on the instruments with occasional assistance from the Core Director and/or staff. Completion of the Analyzer Class by itself does not mean an individual is certified to run the instruments without supervision. 'Unassisted user' status requires demonstration of independent ability as determined only by the Core Director or staff.  Simply taking the class does not constitute one being an unassisted user.   After completion of the class, the user must demonstrate proficiency to the flow core director or staff on the instruments before being considered an *unassisted* user.  

Due to the complex nature of sorters, training on the AriaII is not normally available unless certain conditions are met.   Sorters 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 Flow Core Director.

We recommend that all users review our educational materials on the principles of flow cytometry, as well as other on-line 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. These are two necessary steps needed for the successful understanding and operation of the instruments. 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, and 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. (Procedures to follow below).

Users are encouraged to discuss experimental design with the Facility Manager, particularly if multiple fluorochromes are employed.


Operational Procedures

We encourage investigators to run their own samples on the FACS Fortessa and FACS LSRII, as this gives the investigator a better understanding and more control of their experiments, data, and the limitations of the technology.   However, individuals wanting to run their own samples need to be trained to run these instruments prior to running their own samples.  Please speak with the Facility Manager to arrange training.  A fee-for-service "Analyzer Class" is available for the FACS Fortessa and LSRII, please see above under "Training".   

 


Booking

All machines must be booked in advance. Booking is done via the Outlook Calendar and MS Outlook must be installed on a computer before one can book time on any of the four machines. Booking from TMHRI webmail is not available.

If one plans to use the instruments on a regular basis, it is best that one add the appropriate machine to their shared calendars. To do this, first contact the Flow Core Director and request access to the Machine Specific Calendars.   A PDF file will be sent to you to complete the second part where 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

In booking 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/required.  ALWAYS include your contact information in your booking.  


Booking the FACS Fortessa/LSRII

Limitation and etiquette:

  1. Instruments may not be booked for more than 3 hour blocks. Booking consecutive blocks (6 hours) may only be done with the Flow Cytometry Core Director's prior approval.  This is to prevent monopolization of the instruments. Users are expected to be considerate of the other users.
  2. Users must book the time the procedure is expected to take. If the estimated time grossly underestimates the actual experimental time, the Flow Core Director reserves the right to truncate the experiment if someone else has signed up afterward. In such a case, the User that is running overtime must consult with the User signed up afterward. In addition, booking more time than is needed is an equally unacceptable practice.
  3. 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 Core Director.
  4. 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 Core Director when conducting a kinetic or time course assay. Assays expected to take longer than 3 hours should be booked in the late afternoon to be considerate of other users.
  5. Complete contact information must also be provided in case your booking needs to be moved or adjusted by the Core 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 key to keeping the appointment. However, certain situations may alter the arrangement between the User and operator.

  1. 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 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.
  2. 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.
  3. 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, i.e., bulk sort versus a sort into plates.
  4. Complete contact information must also be provided in case the booking needs to be moved or adjusted by the Facility Manager. Please see the figure below for booking details.

All users must have the following information in their bookings whether it be for analysis or sorting.   If one is doing analysis only, one doesn't need to worry about sorting conditions.  


Machine specific protocols

These protocols only cover user-operable instruments, specifically the FACS Fortessa and LSRII. The Core Director and staff are responsible for the Q/C and monthly cleaning of all the machines. This involves swapping the sheath tank for various cleaning solutions, which takes upwards of three hours and removes the machine from service temporarily. However, 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.

REQUIRED PROCEDURES

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/LSR II (Shut-down Procedure)

•  Run a tube with 2 ml of 10% 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).

*DO NOT LEAVE MORE THAT 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 Core Director and staff.  Requests to run a sample containing a known or potential biohazard must be given, in writing, to the Core Director for review by the institutional Laboratory Operations Safety Officer and Flow Core Director. 

The written statement must include the following information: 

  1. What is the aim of the project?
  2. What is the nature of the cells in question (species, tissue source, etc.)?
  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 Data 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 back-up 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-R's, or DVD+R's.

All Flow Cytometry related computers have a second second (Drive D) storage drive 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.