NATIONAL CANCER INSTITUTE - CANCER.GOV

Contact Information

Primary Contact

Ferenc Livak, Ph.D.
Facility Manager

Location

37 Convent Drive
Building 37, Rooms 6008 and 6011
Bethesda, MD 20814

Additional Contacts

Karen Wolcott
Senior Research Assistant
Subhadra Banerjee, PhD
Senior Research Assistant

Overview

Flow Cytometry Core (LGI) offers established technologies to support studies using flow cytometry and cell sorting.

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 labeling 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.

Keywords

405488561635)639)640)FACSFACS AriaFACS Aria (SORP) 3 lasers (355FACS Aria (standard) 3 lasers (405FACS Core Laboratory (LGI)FACSCaliburFACSCalibur 2 lasers (488LSR FortessaLSR Fortessa 4 lasers (355LSRIILSRII 5 lasers (355MoFlo AstriosMoFlo Astrios 5 lasers (355cell sortingFlow Cytometryflow cytometrynci-core