FLUORESCENCE ACTIVATED CELL SORTING FACILITY     The Fluorescence Activated Cell Sorting Facility contains a laser activated, state-of-the-art cell analysis and sorting machine; the BD FACSVantage SE with CloneCyt Plus, DiVa and TurboSort options is one of the most advanced flow cytometers in the world. IN addition, it has two analysis machines: a nine colour (eleven parameter) Dako Cyan flow cytometer and a BD FACSCalibur BD FACSVantage SE CELL SORTER The BD FACSVantage SE System is equipped with an air-cooled Spectra-Physics HeNe Laser (633nm) and a water cooled Coherent Enterprise II laser. The Enterprise II is a small frame ion laser that has multiple wavelength outputs, providing simultaneous lines at 488nm and 351-364nm.      The BD FACSVantage SE System is able to collect 2 scatter and 8 fluorescent signals per cell and can achieve sort speeds of up to 7,000 cells per second on high speed and up to 25,000 cells per second using the BD TurboSort option in analogue mode. The FACSDiVa option provides zero dead time, allowing faster sorts (up to 60,000 events per second) and higher yields than the standard Vantage SE, and enables 4-way sorting with the QuadraSort functionality. The DiVa option uses matrix algebra for fully independent compensation of all channels. Pulse processing allows the measurement of area, width and ratio of detector pulses, and can be used to detect doublets in DNA analysis or the ratio of two fluorescence signals for use in calcium flux measurements.    The BD CloneCyt Plus option is able to deposit a predefined number of cells into a wide variety of collection devices such as microscope slides, 96 or 24 well plates. In addition, BD IndexSort™ Technology allows specific information regarding single cells sorted into each well of a 96-well tray to be recorded and linked to clonal expansion. There are many applications for this type of technology, for example, monoclonal antibody production, isolation of gene transduced cells to study gene expression, and PCR at the single cell level. DAKO CyAn ADP FLOW CYTOMETER    In 2006, the CGC acquired a nine colour flow cytometric analysis machine, the Dako Cyan flow cytometer. Equipped with three lasers, with excitation wavelengths of 488 nm, 635 nm and 405 nm, the Cyan features a high signal strength solid optical flow cell, full interlaser compensation and analysis rates of up to 50,000 events per second.

BD FACSCalibur FLOW CYTOMETER     The FACSCalibur is equipped with a 488 argon laser and can measure up to three fluorescent colors, as well as FSC and SSC. The FACSCalibur is engineered with fixed optical, electronic and fluidic components, giving it great stability and ease of use. FLUORESCENCE MICROSCOPE     The Fluorescence Activated Cell Sorting Facility also houses a range of other equipment to facilitate the preparation and analysis of cell samples. The Center's fluorescence microscope is an Olympus BX51 inverted microscope with dual photomicroscopy options: the PM-30 automatic photomicrographic system for film based images and the Optronics MagnaFire digital camera for computer based imaging. Viewing options include brightfield, reflected fluorescence, phase contrast (three modes) and DIC (Nomarski). The filter blocks available allow visualisation of FITC, PE, DAPI, Texas Red, Hoechst, PI, Fluo-3, Rhodamine Dil and Ethidium Bromide. The objectives available are: CFI Plan Fluor 4x and Dll 10x, 20x, 40x and 100x.

CELL COUNTER    A dual threshold Beckman Coulter Z1 cell counter is used to count total blood cell and lymphocyte numbers in blood or experimental samples. Based on the principle of electrolyte impedence, the counter can be used to count leukocyte numbers without plasma membrane lysis.     A Shandon Cyrotome E Electic Cryostat is available for the preparation of frozen sections

LIGHT PATH CONFIGURATION OF FACS DIVA CELL SORTER     Relative Fluorochrome Intensity on Facs Vantage:   Brightest PE,  PE-Cy7,  PE-Cy5  Intermediate APC-Cy7, APC, FITC, TxRed-PE Weakest PerCP Useless PerCP-Cy5.5

Laser Excitation Channel Fluorochrome Emission  Filter Enterprise II 488 nm FL1 FITC 525nm 530/30       GFP 540nm 530/30       CFSE 517nm 530/30       Alexa Fluor 488 550nm 530/30       Acridine Orange DNA: 530nm  530/30      FL2 PE 575nm 575/26     FL3 PE-Cy5 670nm 661/37       PerCP 677nm 675/20       PE-Cy5 670nm 661/37       PE-Cy5.5 695nm 710/50       PE-Cy7 760nm 740LP       PerCP-Cy5 670nm 675/20       PerCP-Cy5.5 695nm 712/21       7-AAD 660nm 675/20       Acridine Orange  RNA: 650nm 660/20     FL4 TxRed/PE 615nm 630/22       PI 620nm 610/20 HeNe 633 nm FL5 APC-Cy7 760nm 785/50     FL6 APC 660nm 660/20       PerCP 677nm 675/20        Cy5 670nm 660/20       PerCP-Cy5 670nm 675/20       Alexa Fluor 633 639nm 630/22 Enterprise II 350 nm FL7 Indo 1 Ca++: 408nm 405/20       Marina Blue 460nm 424/44       DAPI 461nm 424/44       Hoechst 33342 450nm 424/44       Cascade Blue 423nm 424/44       AMCA 350 445nm 424/44       Alexa Fluor 350 445nm 424/44     FL8 Indo 1 No Ca: 485nm 485/22       Hoechst 33258 478nm 485/22       Blue FP 490nm 485/22 Default configuration

LIGHT PATH CONFIGURATION OF FACSCALIBUR CELL ANALYSER

Excitation Channel Fluorochrome Emission  Filter 488 nm FL1 FITC 525nm 530/30     GFP 540nm 530/30     CFSE 517nm 530/30     Alexa Fluor 488 550nm 530/30     Acridine Orange DNA: 530nm  530/30    FL2 PE 575nm 585/42   FL3 PE-Cy5 670nm 650LP     PerCP 677nm 650LP     PE-Cy5 670nm 650LP     PE-Cy5.5 695nm 650LP     PE-Cy7 760nm 650LP     PerCP-Cy5 670nm 650LP     PerCP-Cy5.5 695nm 650LP     7-AAD 660nm 650LP     Acridine Orange  RNA: 650nm 650LP

LIGHT PATH CONFIGURATION OF CYAN CELL ANALYSER

Excitation Channel Fluorochrome 488nm FL1 FITC     GFP   FL2 PE   FL3 PE-TxRed     PI   FL4 PE-Cy5     PerCP     7AAD   FL5 PE-Cy7 635 nm FL6 CAS B     CFP     DAPI   FL7 CAS Y 405 nm FL8 APC   FL9 APC-Cy7

PROTOCOLS BD application notes Fluorochromes (Salk) Fluorochromes (NCI)  Fluorochrome conjugation protocols Cell surface staining Intracellular cytokine staining CFSE staining  Software A perspective on compensation MANUFACTURERS BD Caltag Glen Spectra  Molecular Probes  Renovar  OTHER LINKS: History of the cell sorter Comparative Genomics Centre home page Comparative Genomics Centre News James Cook University home page

Comparative Genomics Centre, James Cook University, Key words: Autoimmune diabetes, Type 1 diabetes mellitus, childhood diabetes, lupus, systemic lupus erythematosus, haemolytic anaemia, hemolytic anemia, Coombs' test, antinuclear antibodies, renal failure, glomerulonephritis, gastritis, type A gastritis, pernicious anemia.