Laboratory of Genome Integrity (LGI): Flow Cytometry Core

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)