Institute of Immunology & Infection Research

Flow Cytometry Core Facility


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FACSAria(BD) High-Speed Digital Cell Sorter

- triple laser 407nm Violet, 488nm Sapphire and 633 HeNe
- High-performance sorting
- Accudrop drop-delay optimisation
- 11 parameter
- 9 colour analysis


Performance  (Top)
Fluorescence Sensitivity Estimated detection limit is 125 molecules of equivalent soluble Fluorescein and Phycoerythrin 
Fluorescence Resolution Coefficient of variation PI-Area of < 3%, full peak for propidium iodide–stained chicken erythrocyte nuclei

Coefficient of variation Hoechst-Area of < 3.5%, full peak for Hoechst–stained chicken erythrocyte nuclei
Forward and Side Scatter Sensitivity Sensitivity enables the separation of fixed platelets from noise, identification of bacteria and 0.5-micron beads
Forward and Side
Scatter Resolution
Scatter performance is optimized for resolving lymphocytes, monocytes, and granulocytes
Acquisition rate Maximum acquisition rate 70,000 events/second
At 70psi and 90kHz an average threshold rate of 25,000 events/second for a 4-way sort can result in a purity >98% and a yield >80% of Poissson's expected yield
Viability Dependant on cell type - sorts of cell lines and PBMC resulted in viable cells capable of extended proliferative ability
Sort Collection Devices 2-way sorting - microtubes, 12x75mm and 15ml tubes
4-way sorting - microtubes and 12x75mm tubes
Automatic Cell deposition Unit allows for slides, 6,24,48, 96 and 384 well plates
Temperature control Sample input step control : 4, 20, 37 and 42 oC
Collection control : 4 - 42 oC water recirculation

Excitation Optics  (Top)
Optical Platform Fixed-alignment cuvette flow cell
Lasers 10mW  407nm,  Point Source Violet solid state laser
13mW  488nm,  Coherent Sapphire solid state laser
11mW  633nm,  JDS Uniphase HeNe air-cooled laser

Emission Optics  (Top)
Optical Coupling Quartz cuvette flow cell is gel-coupled  by refractive index matching optical gel to the fluorescent objective lens for optimum collection efficiency
Background Rejection Obscuration blade and slit minimize unwanted laser radiation at the detector
Forward Scatter Detector and Filter Photodiode detector with 488/10 bandpass filter
Side Scatter Detector Photomultiplier with 488/10 bandpass
488nm
Fluorescence Detectors and Filters

530/30nm     FITC /Alexa-488 / GFP / CFSE
576/26nm     PE / DsRed
610/20nm     PE-Texas Red / PI
675/20nm    PerCP / PE-Cy5 / 7AAD
780/60nm    PE-Cy7 
633nm
Fluorescence Detectors and Filters
660/20nm   APC / Cy5 / Alexa-647
780/60nm   APC-Cy7 / APC-Alexa-750

407nm
Fluorescence Detectors and Filters
530/30nm   Alexa-430 / CFP
450/40nm   Pacific Blue / DAPI / Hoechst /                        Vybrant Violet  

Fluidics  (Top)
General Operation Fluidics cart provides sheath and cleaning fluids to the instrument and receives waste.
Sheath pressure is adjustable from 2 to 75 psi.
Fluid Reservoirs 10-L sheath and waste containers provided.
5-L cleaning reservoirs provided.
Autoclavable containers and fluidics probes.
Accommodates BD FACSFlowTM sheath fluid
containers.
Sample Flow Rates Adjustable sample pressures from 1 to 11 (0.2 to 1.2
psi over the sheath pressure).
Flow Cell Quartz Cuvette 160-um x 250-um, 15-mm long
Sample Concentration Single-cell suspension of 105 to 2 x 107 particles/mL recommended range

Signal Processing  (Top)
Workstation Resolution 18-bit data acquisition
262,144 linear channels
Dynamic Range Bi-expontial plots and  provide five log decade display
Fluorescence Compensation Networks No limit to inter- and intra-beam compensation
Pulse Processing Width and area measurements available for all fluorescence parameters.
Ratio measurements for intra-laser parameterss
Time Time available correlated to any parameter

Data Management System   (Top)
Workstation PC 
Central Processing Unit (CPU) Pentium4 @ 3.0GHz
Memory > 2 GB RAM
Data Storage 40 GB and 160 GB hard drive
CD/DVD 16x DVD +/- RW, Dual Layer
Monitor 2x19-inch MultiSync Monitor
Data File Structure Flow Cytometry Standard (FCS) 3.0/2.0
ASCII results file for data export