Established Technologies

• Cell sorting of both infectious(HIV, SIV, HTLV) and non-infectious samples
• Multi-color immunophenotyping
• Cell cycle analysis
• Proliferation analysis (Ki67, PCNA, BrdU, CFSE)
• Apoptosis measurement (Annexin V, JC1, Active Caspase 3, TUNEL)
• Intracellular cytokine analysis
• Stem cell analysis and sorting
• Calcium flux analysis
• Bead array analysis of soluble proteins
• Phosphorylated protein analysis
• Fluorescent protein analysis and sorting
• Quantitative flow cytometry
• AutoMACS Pro magnetic bead separator

Developing Technologies

Instrumentation

• 28-color BD FACSymphony A5 – 355nm, 405nm, 488nm, 532nm, and 628nm lasers
• 28-color BD FACSymphony A5 – 355nm, 405nm, 488nm, 532nm, and 628nm lasers
• 28-color BD FACSymphony A5 – 355nm, 405nm, 488nm, 532nm, and 628nm lasers
• 18-color BD FACSAriaIIIu cell sorter – 405nm, 488nm, 561nm, and 640nm lasers
• Miltenyi autoMACS Pro magnetic bead separator

User Guidelines

The VB FACS Core Facility is open 24 hours/day, 7 days/week. Access to the facility is controlled by proximity card readers that respond to NIH ID badges. Users are granted access following completion of training on the instruments.

The core facility is staffed from 8:00 AM to 6:00 PM Monday-Friday. After hours telephone support by the Facility Manager is available 7 days/week

Instrumentation

• FACSCanto I – 2 lasers (488, 633)
• FACSCanto II – 3 lasers (405, 488, 633)
• LSR-Fortessa – 4 lasers, 16 filters

Established Technologies

• Immunophenotyping (up to 15-color)
• Specialized immunophenotyping for clinical trial monitoring – Regulated testing under the Clinical Laboratory Improvement Amendments (CLIA) available for CD3, CD4, CD8, NK, CD19+ B cells
• Intracellular markers, including cytokines
• Proliferation analysis (Ki67)
• Detection of fluorescent reporter GFP
• Dedicated program courier support for transport of samples to/from NCI-Frederick

User Guidelines

The Clinical Support Laboratory is open to all NCI intramural clinical investigators. Project scope is defined in consultation with the customer. Cost estimates based on actual time and materials are provided for investigator review/approval before any project is initiated.

To request services from this CCR core facility, you must submit your requisition through NAS.

Typical Assays Performed with Core Lab Instruments

• Immunophenotyping of mouse and human cells
• Apoptosis assays, including Annexin V, TUNEL, caspase activation or mitochondrial membrane potential
• Cell cycle (DNA content) analysis
• Cell health and proliferation monitoring
• Sterile Cell sorting (physical separation) for bulk collection or deposition in 96- or 384-well plates
• Sorting and analysis of cells by fluorescent reporters such as GFP, BFP, mCherry, etc.

Instrumentation

• BD FACS Symphony S6 cell sorter in BSL2* lab with 5 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm, UV/350nm).
• BD FACSAria II SORP cell sorter in BSL2* lab with 4 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm).
• BD FACSAria II cell sorter with 5 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm, UV/350nm).
• BD FACSymphony A5 analyzer with 5 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm, UV/350nm).
• BD LSRII SORP analyzer with 4 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm).
• BD LSRII analyzer with 3 lasers (blue/488nm, red/640nm, violet/405nm).
• BD Canto II analyzer with 3 lasers (blue/488nm, red/640nm, violet/405nm).
• BD LSRII Fortessa analyzer with 4 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm).
• Miltenyi Biotec MACSQuant-16 analyzer with 3 lasers (blue/488nm, red/640nm, violet/405nm) and optional 96-well sampling.
• Cytek Aurora Spectral Cytometer with 5 lasers (blue/488nm, red/640nm, violet/405nm, yellow-green/561nm, UV/350nm).

User Guidelines

The LCIM CCR-Frederick Flow Cytometry Core Facility maintains instrumentation, and provides cytometry training, analysis and sorting services to CCR investigators. Services are made available to Investigators outside of CCR on a case-by-case basis. Our fee structure is in the table below.

Trained, certified users may run their own samples on the analyzer instruments; staff operates cell sorter instruments.

To request services from this CCR core facility, please contact Jeff Carrell or Megan Karwan by email or phone (301-846-5811).

Instrumentation

• 2 FACSCaliburs – 2 lasers (488, 635)
• LSRII – 5 lasers (355, 405, 488, 561, 635)
• LSR Fortessa – 4 lasers (355, 405, 488, 639)
• FACS Aria (standard) – 3 lasers (405, 488, 639)
• FACS Aria (SORP) – 3 lasers (355, 488, 639)
• MoFlo Astrios – 5 lasers (355, 405, 488, 561, 640)

Established Technologies

Applications that run on FACS Caliburs include:

• Immunophenotyping (up to 4-color)
• Intracellular markers, including cytokines and phosphoproteins
• Cell cycle analysis using propidium iodide for mammalian cells and cytox green for yeast
• Cell cycle-associated BRDU labelling of S-phase cells and phosphohistone H3 labeling of mitotic cells
• Apoptosis by annexin V binding, TUNEL, caspase, or FLICA
• Detection of fluorescent reporters GFP, YFP, and dsRed
• Membrane potential with oxonol and cyanine dyes
• Mitochondrial membrane potential with JC-1, Mito Tracker Dyes, oxidative activity using DCFH-DH and DHR
• Lipid probes
• Aldefluor

Applications used on the LSRs include:

• Immunophenotyping using up to 10 simultaneous probes (including CFSE)
• SP cells (side population)
• RFP (red fluorescent proteins, including cherry and tomato)
• Qdots
• Cell cycle analysis with the UV-excitable dyes Hoechst 33342 and DAPI on both mammalian cells and bacteria
• Simultaneous analysis of cell cycle (HO3342) and GFP in viable cells
• HO/Pyronin for DNA/RNA
• Intracellular calcium ratiometric measurements using indo-1

Cell Sorting:

• Subsets of murine T, B, and stem cells (including SP cells) for in vitro culturing and functional assays, re-injection into the mouse, or preparation of RNA, DNA, or protein
• Primary murine keratinocytes, melanocytes, thryroid, lung, liver, prostate, and mammary cells – sorted by expression of specific antigens
• Sorting of tumor xenografts
• Sorting of potential cancer stem cells from cell lines and primary tumors based on surface antigen expression, SP (side population), or aldeyde dehydrogenase
• Cell lines transfected with GFP, YFP, or RFP reporters
• Preparation of high-luciferase cell lines for imaging based on co-expression with GFP or RFP
• Cell cycle compartment sorting using viable cells stained with Hoechst 33342 for characterization of cell cycle-related proteins
• Plate-sorting for single cell PCR
• Slide-sorting for single cell PCR (Advalytix)

User Guidelines

The Flow Cytometry Core is open to CCR investigators who do not have their own flow cytometry facilities, with preference given to Bldg. 37 scientists. An annual fee, as yet to be determined, will be charged to all principal investigators whose staff uses the lab.

Following successful individualized training on the bench-top flow cytometers, a user will run his/her own experiments and may access the flow cytometers on a 24/7 basis.

After individual consultation, cell sorting is done by the core facility staff on a scheduled basis.