Flow Cytometry Policies
To ensure a productive, safe, and collaborative environment, all users of the Flow Cytometry Core are expected to follow the policies outlined below. Please contact the facility with any questions prior to your appointment.
1. Instrument Access & Scheduling
- Unassisted sessions on the BD LSRFortessa X-20 Analyzer can be scheduled via CIDER or by contacting the facility directly through email.
- Assisted sessions on either the BD LSRFortessa X-20 or the Cytek Aurora CS must be scheduled via the Project Request Form or email.
- For assisted appointments, users are asked to provide control samples, details about experimental design, and information on all fluorophores used
- Assisted sort sessions are reserved and managed by Dr. DelVichio and are billed according to actual usage time.
2. Training
BD LSRFortessa X-20 Analyzer
- Independent user status is granted following the successful completion of flow cytometry training with Dr. DelVichio.
- Training includes approximately 2.5 hours of instruction covering:
- Core principles of flow cytometry
- Instrument operation and software use
- Best practices for data acquisition
- Following completion, users are required to schedule an assisted session before gaining full independent access.
Cytek Aurora CS
- Independent operation of the Cytek Aurora CS requires specialized training in cell sorting.
- The initial training session is approximately 4 hours and includes:
- Fundamental flow cytometry principles
- Introduction to cell sorting workflows
- Instrument and software operation
- After training, users must complete 10 hours of assisted use, during which proficiency will be assessed.
- Upon review, independent access may be granted. If additional support is required, further training may be scheduled at the discretion of facility staff.
3. Data Storage and Management
- The facility provides temporary data storage through the BD Backup Drive.
- Long-term storage is the responsibility of the user. Please ensure you download and back up your data promptly.
- Data remaining on instrument workstations will be automatically and permanently deleted after two (2) months.
4. Instrument Cleaning Responsibility
- Cleaning the Fortessa X-20 after use is mandatory and takes approximately 18 minutes to complete.
- This is the responsibility of the user. Failure to follow the cleaning protocol may result in mandatory retraining, billed to the user's lab.
- Please schedule enough time within your reservation to both complete your experiment and clean the instrument.
5. Use of New Specimens and Biohazards
- If your experiment involves a new specimen type or material, please consult with Dr. DelVichio before your session.
- The Cytek Aurora Cell Sorter is housed within an aerosol containment unit certified by Environmental Health and Safety (EHS). This allows the instrument to safely accommodate biohazardous samples when properly reviewed and approved.
6. Acknowledgment in Publications
- Publications resulting from data acquired through the Core should include an acknowledgment of the facility.
- In certain cases, co-authorship may be appropriate, particularly when core personnel contribute significantly to experimental design, data acquisition, or analysis.
- For guidance, please refer to the ABRF Authorship Guidelines.
Cell Sorting Guidelines
To ensure the success and efficiency of your cell sorting appointment, please review and adhere to the following guidelines. These policies are in place to maintain the integrity of your samples and the performance of the instrumentation.
1. Scheduling a Sort
- Researchers must contact the facility or complete the [Sort Request Form] to schedule a sort or consultation.
- Sorting requests should be submitted at least three (3) business days in advance of the desired appointment.
- While we aim to accommodate all requests, short-notice scheduling is subject to availability and cannot be guaranteed.
2. Instrument Capabilities
- The sorter supports the detection of up to 64 fluorescent parameters from a single sample.
- Up to six (6) distinct populations may be sorted per run.
- Sorting can be performed into collection tubes or multi-well plates, depending on experimental requirements.
3. Sample Preparation Requirements
- Samples must be provided as single-cell suspensions. Clumped or aggregated samples will compromise sort accuracy and yield.
- Samples must be filtered prior to arrival at the facility. We recommend the use of FACS tubes (5 mL round-bottom polystyrene tubes with a 35 µm cell strainer cap). FACS tubes are available for purchase at BioSupply.
- Do not prepare samples in media containing phenol red, as it can interfere with fluorescence detection. Preferred buffers include PBS or HBSS, free of Ca²⁺ and Mg²⁺, supplemented with no more than 2% FBS or BSA.
4. Required Controls and Reagents
- Please bring all required controls, including:
- Unstained controls
- Single-stained reference controls
- Fluorescence Minus One (FMO) controls
- Experimental samples
- The facility is not responsible for reduced sort purity due to missing or inadequate controls.
- Researchers are responsible for providing appropriate collection tubes or plates and collection media at the time of the sort.
5. Collection Buffer Recommendations
- Collection tubes should be pre-filled to approximately 1/3 of their volume with a suitable collection solution.
- Common collection media include:
- PBS with 50% FBS
- Cell culture media with 50% FBS
- 100% FBS
- Optional: Pre-coating collection tubes with 1% BSA overnight may improve post-sort cell recovery.
- Sheath fluid used in the sorter is 1X PBS.
6. Cell Count Requirements
- A minimum of 500,000 viable cells is recommended for sorting.
- If fewer cells are available, we will work with you to achieve the best possible outcome; however, yield may be limited.
7. Nozzle and Sort Configuration
- The instrument utilizes a droplet-based sorting mechanism with interchangeable nozzles.
- Nozzle size selection depends on cell size and fragility. Please consult with facility staff in advance to determine the optimal configuration for your cell type.
8. Arrival Time and Setup
- Instrument setup requires approximately 45 minutes prior to the start of each sort.
- Please arrive on time for your scheduled appointment. If you anticipate a delay, notify the facility as soon as possible so that adjustments can be made to the setup timeline.