The NCI, CCR, LCBG Flow Cytometry Core Facility has provided service to 78 Principle Investigators and 223 users from 31 NCI labs or branches from January to August 2013. The flow cytometry cell analyzers were used a total of 1,803 hours; FACS Caliburs for 387 hours, LSRII for 558 hours, and the LSRFortessa for 858 hours. During the same time, flow cytometry cell sorters were used a total of 1,370 hours; FACSAria-UV for 553 hours, FACSAria-Violet for 636 hours and the MoFlo Astrios for 181 hours. Identifying and studying cancer stem cells are one of the major research areas of the Flow Cytometry Core Facility users. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic and lung carcinomas. The LSRII flow cytometer, the special order FACSAria, and the MoFlo Astrios cell sorter are frequently used for these assays. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of labs using the Flow Cytometry Core Facility. The core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with any of the green, yellow, blue and red fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have then been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of luciferase. These cell lines have then been used to establish tumors in mice and to image metastasis. Flow cytometry is also frequently used for looking at cell growth (cell cycle analysis) and mechanisms of cell death (apoptosis) to examine actions of cancer drugs. The Flow Cytometry Core Facility has implemented a new policy for cell sorting. According to the recently developed NIH Policy for Biosafety of Cell Sorters (July 28, 2012) sorting of human cell lines, human cells and 2nd or 3rd generation Lentivirus in human cells require the sort to be performed at a BSL-2 with enhanced precautions biosafety level. The Beckman Coulter MoFlo Astrios cell sorter has recently been upgraded with a custom designed SterilGARD Class II Biosafety Cabinet with an onboard HEPA filtration system that is uniquely configured to fit on the instrument bench to provide safe, effective aerosol evacuation. This instrument upgrade enables the flow cytometry staff to perform sorts in compliance with the new guidelines. A second Baker Class II Biosafety Cabinet has been ordered and will house a BD FACSAria in the future. Additionally, plans to renovate the facility to provide a separate space for the MoFlo Astrios and a FACSAria so that sorts requiring this biosafety level can be done every day rather than only two times a week. Currently, core staff restrict entry to the facility and use powered air purifying respirators (PAPR's) on Tuesday and Thursday to preform sorts of this kind. Appointments to use the analyzers are scheduled with shared Microsoft Outlook calendars. The cell sorters are scheduled after the NCI, CCR, LCBG Flow Cytometry Core Facility Biosafety Questionnaire and Sort Request form has been submitted and reviewed by the Core Manager. The core website, located at https://labshare.nih.gov/nci/ccr-facs is currently under construction. It will soon contain pertinent information about the services the core offers as well as provide an easy scheduling system.