Flow Cytometry Shared Resource | UC Davis Comprehensive Cancer Center

Analytic cytometers biohazard policy:

• Operators must fix samples run on benchtop analytic cytometers.

Appointment policies:

• All instrument usage time requires a scheduled appointment.
• Analytic cytometers: Operator-assisted appointments require a minimum of 24 hours advance notice.
• Approved, trained users may make same-day, self-operation appointments.
• Cell sorting appointments require a minimum of 24 hours advance notice.
• Cell sorting requires operator assistance.
• Evening and weekend appointments are for trained, self-operated use only. 

 Appointment duration policies:

• Cell sorters and 5-laser Fortessa analytic cytometer: 1-hour minimum
• Other analytic cytometers: 30-minute minimum

 Decontamination and cleaning of fluidics policies:

• All users must wash the fluidics for a minimum of 15 minutes after using the cytometer; HTS users must add additional time for HTS cleaning.
• Post-acquisition washing and cleaning procedures count as normal run time. The FCSR bills them as part of the total appointment time.
• Proper decontamination and cleaning of fluidics tubing and flow cell is required after each use of the cytometer.

 Training session policies:

• Laboratory personnel determine an investigator’s ability to independently operate cytometers.
• Retraining: Users earn self-operation privileges, subject to laboratory management approval. We will schedule retraining as necessary at the discretion of FCSR staff. We bill retraining as assisted time.
• Training sessions require a minimum of two hours, charged at the assisted user rate. Users must supply a sample for training purposes.

The procedures below ensure safe, efficient and equitable facility operations. Any violation of these procedures may result in termination of access privileges.

Biohazards:

Two FCSR instruments (the Astrios EQ in Tupper Hall and the BD InFlux Cell Sorter in the Institute for Regenerative Cures) work with biohazards. Please read our policies on using biohazards:

• Users must notify their intent to use biohazards to the laboratory manager before we approve your application.
• You must notify facility personnel of potential biohazards prior to scheduling appointments and running samples.
• All experiments must comply with UC Davis regulations for biosafety. All samples run on benchtop analyzers must be fixed.
• Users must take all tubes and samples left after analysis back to their home laboratory in containers approved for safe transport.
• UC Davis Environmental Health and Safety must approve an amendment before a user sorts biohazards.
• The FCSR will terminate access for any users who fail to meet the facility biohazard requirements.

Operators and self-operation:

The FCSR has several analytic (non-sorting) cytometers available in Davis and Sacramento for operation by trained personnel. We also offer training for self-operation using samples provided by the investigator. This is by appointment only. Laboratory personnel determine when an investigator can self-operate equipment. We can provide keys to the facility for after-hours use.

Sorter self-operation is a major commitment. We recommend self-operation only if the user intends to sort cells for a significant period of time. The facility manager may allow self-operation of the BD FACS Aria II cell sorter in Sacramento when staff is available to assist. To self-operate the Aria sorter, we require training with facility staff followed by hands-on experience until users acquire sufficient skill.

Please review these self-operator policies:

• We expect users to master normal operations without a facility operator’s assistance. These include startup, sorting, analysis, sterilization, maintenance and shutdown.
• Self-operators must negotiate with the facility operator for help when independently operating equipment. Note that extensive reliance on facility operator for assistance will lead to full charges.
• Self-operators can sign up for 15 minutes of facility operator time and self-operate for the remaining time.
• Equipment abuse may result in self-operator privilege curtailment until problems are clarified.

Instrument scheduling:

• The FCSR schedules operator-assisted runs Monday through Friday, 10 a.m. to 4 p.m. Users may negotiate for after-hours, assisted-use times. Please sign up on the instrument scheduling calendar.
• Users who sign up for assisted use must ensure a staff member is available for assistance at that time. Users may schedule an instrument by selecting “Instrument Scheduling” from the menu.
• You must provide 24-hour notice for cancellation of a scheduled run to avoid charges. If signup times shift or the facility operator makes an adjustment, we may waive a cancelation charge.

Equipment running time:

• A signup entitles the user to the specified time slot only. This strict schedule interpretation enables users and operators to meet obligations. A user with a reservation may terminate a previous user's unfinished run.
• If equipment malfunctions or delays prevent usage, the user may request a reduction in charges. The FCSR bills for actual instrument use time (including the minimum described below).
• The FCSR bills users for extra time when more than an hour of reserved slot time remains unused.

Facility running time:

• Most machines require a 30-minute minimum reservation time. The Fortessa 5-laser cytometer and the cell sorters require one hour.
• Extra charges apply for runs that extend beyond the signup time. The FCSR charges for reservations cancelled less than 24 hours in advance.
• Users must notify the facility operator of equipment malfunctions or issues. We will adjust a user’s total running time when delays occur from malfunction or previous users.
• Nozzle clog delays may result from clumpy samples or excessive boost duration. They are considered chargeable time. If a previous user left a clog or clearing the flow cell requires extra time, we will adjust the running time.

Facility rates:

• The Department of Medical Pathology bills accounts each month.
• Please direct billing questions to Bridget McLaughlin.
• You must provide a DaFIS recharge number to use the facility.

Data archival:

• Facility users are responsible for all data archival.

Problems:

• Users should immediately report problems at the facility.
• For instrumentation problems, contact Bridget McLaughlin.
• Director Barbara Shacklett is the final arbiter for problems that arise at the facility.

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• Givan, AL. Flow Cytometry: First Principles. New York: Wiley-Liss;1992.
• Maecker HT, Frey T, Nomura LE, Trotter J. Selecting Fluorochrome Conjugates for Maximum Sensitivity Cytometry. Cytometry A. 2004;62(2):169-73. 
• Diamond R, DeMaggio S, eds. In Living Color: Protocols in Flow Cytometry and Cell Sorting. Springer-Verlag; 2000.
• Park LM, Lannigan J, Jaimes MC. OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. Cytometry Part A, 2020;97(10):1044-1051.
• Roederer M, Darzynkiewicz Z, Parks DR. Guidelines for the presentation of flow cytometric data. Methods Cell Biol. 2004;75:241-56.
• Tung JW, Parks DR, Moore WA, Herzenberg LA, Herzenberg LA. New approaches to fluorescence compensation and visualization of FACS data. Clin Immunol. 2004 Mar; 110(3): 277-83.

International Society for the Advancement of Cytometry

• Beckman Coulter
• BioLegend
• BD Biosciences
• Bio-Rad
• FluoroFinder
• Novus Biologicals 
• SouthernBiotech
• Thermo Fisher

• Polysciences, Inc.
• Spherotech

• Mario Roederer
• Compensation
• Antibodies

• Current Protocols in Cytometry
• SpectroLearn tutorials from Cytek

Purdue University Cytometry Laboratories

• FlowJo
• DeNovo Software (FCS Express)
• Verity Software House, Inc. (winlist and modfit)