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Fluorescence Activated Cell Sorting Facility

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Fluorescence Activated Cell Sorting Facility
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.

Flow
microscope 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
coulter

 


 

LIGHT PATH CONFIGURATION OF FACS DIVA CELL SORTER

 

Diva Setup

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
Calibur Setup
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
Cyan Setup
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


TABLE OF FLUOROCHROME COMPATIBILITY

FLUOROCHROME ABSORBTION (nm) EXCITATION(nm) EMISSION (nm) FACScalibur FACS DiVa Dako CyAn
Indo-1 330 351 408/485
Marina Blue 365 351 460
DAPI 369 351 451
Hoechst 33342 340 351 460
Cascade Blue 377 351,360,405,407 420
Alexa Fluor 405 401 360,405,407 421
Pacific Blue 410 360,405,407 455
Alexa Fluor 488 495 488 519
FITC 493 488 525
PE 496,565 488 575
PE/TexasRed 496,565 488 613
APC 645 595,633,635,647 660 weak
Cy5 649 633,635 666
Alexa Fluor 647 650 595,633,635,647 668
PE/Cy5 496,565 488 670
PerCP 482 488 675 weak
APC/Cy5.5 650 595,633,635,647 690
PE/Cy5.5 496,565 488 690
PerCP/Cy5.5 482 488 690
Alexa Fluor 700 696 633,635 719
APC/Cy7 650 595,633,635,647 774
bright
PE/Cy7 496,565 488 774 bright
7-AAD 546 488 647
PI 305,540 325,360,488 620

 


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