Flow Cytrometry Core Facility Policies

Users who schedule a block of time are responsible for canceling that reserved time at least 24 hours in advance. If the user does not show up for a reserved appointment, or if the appointment is cancelled less than 24 hours in advance, the user will be billed for the reserved time block.

During both analysis and sorting the FACSAria flow cytometer makes up to 90,000 droplets per second.  When running unfixed cells, many of these droplets may contain viable cells.  While this stream of droplets is routinely caught in a waste receptacle and combined with at least 10% bleach to render the sample harmless, during an instrument failure (such as a partial clog of the nozzle) the potential exists for the production of large amounts of secondary aerosols.  As samples may contain viable cells which may be tumorigenic, contain pathogens and/or toxic/carcinogenic dyes, we all need to be concerned with aerosol containment for the safety of both the facility personnel and the facility users.  In order to understand and minimize the health risks to facility personnel and users the following policies have been initiated:

• To reduce the infectious risk to the instrument operator and the investigator, we require that all samples must be fixed prior to analysis unless prior consent has been obtained from the facility.
• While we do not at this time require HIV, EBV or Hepatitis testing of every sample, we do require that investigators inform the facility if samples are known to be positive.
• In an effort to identify risks associated with each set of samples, investigators must complete the following paperwork:
  • An approved 'Project Biosafety Questionnaire' must be on file in the cytometry core before work on a project is started. This questionnaire must be filled out by the laboratory director (PI) and includes details about each project (i.e. a grant, project or set of experiments). This questionnaire exists in two formats:
    • General Project Biosafety Questionnaire – a two page questionnaire giving details on how the investigator intends to use the facility and identifying potential biosafety risks.  This form should be adequate for the majority of users not doing work with human cells or unfixed samples.
    • Detailed Project Biosafety Questionnaire – a four page questionnaire designed to go into more detail regarding potential biosafety issues.  This form is appropriate to use if your project uses human cells, live cells, or genetically altered cells.
  • A  Daily Cytometry Sample Sheet must accompany each set of samples brought to the flow cytometry core facility. This sheet identifies the PI and investigator associated with the samples, and gives us a contact number if we have questions while running the samples. It also asks you to identify the sample source, whether they have been fixed, whether they have been treated with pharmacological agents, and whether they might be infectious.

We recognize that investigators are already being asked to complete a lot of paperwork, so we have tried to make this as painless as possible.  However it is clear that biosafety risks exist in flow cytometry core facilities, and we feel it is in the best interest of everyone involved to identify, minimize and contain these risks.

Radiolabeled samples may not be used in the facility at this time.

Live cells may be a potential infectious agent themselves, or may be associated with an infectious agent. Therefore fixation is required for all samples prior to analysis in the facility. 

The will be limited exceptions to this rule for:

1. Sorts in which the sorted population will be put back into culture or in vivo, or ?
2. Experiments in which cell viability is being determined in the assay.

All samples (live or fixed) must be accompanied by a Daily Sample Information Sheet which clearly states whether the samples are fixed or unfixed prior to analysis.

Fixatives appropriate for flow cytometry are formaldehyde (1-10% in PBS), paraformaldehyde (1-2% in PBS), and ethanol (70-90% 1 hour or longer).  Due to its high autofluorescence, gluteraldehyde is not a good fixative for flow cytometry.  For samples containing known HIV positive or HIV infected cells, samples must be fixed in 1%-2% formaldehyde for at least 30 minutes prior to flow cytometric analysis.

On the FACSAria, all samples will be run by facility personnel without exception.  On the Yeti we encourage investigators to run their own samples, as we believe this gives the investigator a better understanding and more control of their experiments and data, and also a better understanding of the limitations of the technology.  However, individuals wanting to run their own samples need to be trained to run the Yeti prior to running their own samples.  Training sessions will be held several times each year, or speak with Dr. Perry to arrange a personal training session.

It would be to the benefit of the facility and the Creighton research community in general if the contribution made by the Flow Cytometry Core Facility was acknowledged in publications that include data produced in the facility.  A reprint containing this acknowledgment should also be sent to the facility.  Acknowledgment of the facility will help us to obtain continued outside funding for the facility.