Flow Cytometry

  • Multicolour flow cytometry analysis
  • 5 spatially separated lasers
  • 18 fluorescent detectors
  • Sample loading options (5ml tubes or 96-/384-well plates)

Description

The BD LSRFortessaTM SORP is a benchtop digital flow cytometer. Equipped with 5 spatially separated lasers, 18 fluorescent detectors and 2 light scatter detectors for fast multicolour analysis with accurate data acquisition rate at up to 40,000 events per second (with beads). The BD LSRFortessaTM has a BDTM High Throughput Sampler (HTS) for increased lab productivity by acquiring samples from a 96- or 384-well plates.

Link to the manufacturer: https://www.bdbiosciences.com/en-us

Applications

Multicolour flow cytometry analysis

Specification

Excitation Optics
Laser Wavelength [nm]
355405488561640
Laser Power [mW]
20501005040
Emission Optics
Forward Scatter Detector: photodiode with 488/10 bandpass filter for the 488-nm laser
Side Scatter Detector: photomultipler with a 488/10 bandpass filter for the 488-nm laser

Fluorescence Detectors and Filters:
BPLPND
UV Laser 355
Indo-1 blue530/30505
Indo-1 violet450/50
Violet Laser 405
BV786780/60750
Qdot655670/30630
Qdot605610/20600
Qdot585585/15570
AmCyan525/50505
Pacific Blue450/50
Blue Laser 488
PerCP-Cy5.5710/50685
FITC530/30505
SSC488/10
YellGreen Laser 561
PE-Cy7780/60750
PE-Cy5.5710/50685
PE-Cy5670/30635
PE-Texas Red610/20600
PE586/15
Red Laser 640
APC-Cy7780/60750
Alexa Fluor 700730/45690
APC670/14
Available filters
685/356701.5 in use Aria
675/506351.0 in use Fortessa
670/305952.0
660/205452.0
585/425351.5
560/201
560/401
540/30
424/44
Fluidics - Sample Flow Rates
  • Front key panel provides three modes
    • RUN
    • STANDBY
    • PRIME

  • Continuously adjustable flow rates, plus three preset flow rates
    • LO 12 µL/min
    • MED 35 µL/min
    • HI 60 µL/min
Fluorescence sensitivity
  • FITC : 80 molecules of equivalent soluble fluorochrome (MESF-FITC)
  • PE: 30 molecules of equivalent soluble fluorochrome (MESF-PE)
  • PE-Cy™5: 10 molecules of equivalent soluble fluorochrome (MESF-PE-Cy5)
  • APC: 70 molecules of equivalent soluble fluorochrome (MESF-APC)

FITC and PE measurements performed using SPHERO™ Rainbow Calibration Particles (RCP-30-5A) PE-Cy5 and APC measurements performed using SPHERO Ultra Rainbow Calibration Particles (URCP-38-2K)
Fluorescence resolution
Coefficient of variation (CV) PI: Area, <3.0%, full G0/G1 peak for PI-stained chicken erythrocyte nuclei (CEN)
Fluorescence linearity
Doublet/singlet ratio CEN stained with PI: 1.95–2.05 (488-nm laser)
Forward and Side Scatter Sensitivity
Sensitivity enables separation of fixed platelets from noise, identification of bacteria, and detection of 0.5-µm beads
Forward and Side Scatter Resolution
Scatter performance is optimized for resolving lymphocytes, monocytes, and granulocytes
Signal Processing -
Converter
10-MHz analog-to-digital converter
Signal Processing -
Workstation Resolution
262,144 channels
Signal Processing -
Pulse Processing
Height, area, and width measurements available for any parameter. Ratio measurements are also available
Signal Processing -
Time
Can be correlated to any parameter for kinetic experiments or other applications
BD™ High Throughput Sampler (HTS)
Is available to increase your lab productivity by acquiring samples from a 96- or 384-well microtiter plate.

HTS throughput

Acquisition
  • less than 15 minutes per microtiter plate in high throughput mode using a 2-second acquisition
  • less than 44 minutes in standard mode using a 10-second acquisition

Carryover
  • <0.5% HT mode
  • <0.75% STD mode
  • Fluorescence Activated Cell Sorting
  • 4 spatially separated lasers
  • 16 fluorescence detectors
  • T° control of a sample
    • Sorting on/into device options:
      • 1.5mL tubes
      • 5ml-, 15ml- tubes
      • 6-, 24-, 48-, 96-, 384-well plates
      • Microscope slides

Description

Fluorescence activated cell sorter (FACS) with a quartz cuvette flow cell that is in fixed alignment with the laser, and is gel coupled to the collection optics. BD FACSAriaTM Fusion is equipped with 4 spatially separated lasers, 16 fluorescent detectors and 2 light scatter detectors for fast multicolour analysis and cell sorting with accurate event data acquisition at up to 70,000 events per second. BD FACSAriaTM Fusion is placed in the biosafety cabinet (BSC) Class II Type A2, designed in collaboration with The Baker Company, a leader in biosafety solutions.

Link to the manufacturer: https://www.bdbiosciences.com/en-us

Applications

Flow cytometry analysis and fluorescence activated cell sorting.

Specification

Excitation Optics
Fixed optical alignment of all Class IIIb lasers with the cuvette flow cell. All lasers are solid state and elliptical
  • Beam height: 9 ±3 µm
  • Beam width: 65 ±7 µm
Emission Optics
Forward Scatter Detector: photodiode with 488/10 bandpass filter for the 488-nm laser
Side Scatter Detector: photomultipler with a 488/10 bandpass filter for the 488-nm laser

Fluorescence Detectors and Filters:
BPLP
Violet Laser 405
BV786780/60750
BV711710/50690
Qdot605610/20595
Qdot585586/20550
BV510510/30495
DAPI450/50
Blue Laser 488
PerCP-Cy5.5695/40655
FITC525/20505
SSC488/10
YellGreen Laser 561
PE-Cy7780/60750
PE-Cy5690/20670
PE-Cy5.5670/14630
PE-Texas Red610/20600
PE586/15
Red Laser 640
APC-Cy7780/60735
Alexa Fluor 700705/20685
APC670/30
Fluidics
  • Sheath pressure is adjustable from 5 to 75 psi
  • Sample Flow Rates adjustable dynamic range
  • Nozzles are removable and can be sonicated; available in 3 sizes: 70, 85 and
    100-µm
  • Temperature control of sample input: 4ºC, 20ºC, 37ºC, and 42ºC (adjusted in the software); sample output: from +5°C to +42°C
  • Collection devices: 15ml- and 5ml-tubes, eppendorfs, 6, 24, 96, and 384-well plates. Index sorting can be enabled when sorting single cells. This capability indexes the cell surface phenotype to the well containing that cell
Fluorescence sensitivity
Measurements performed at 70 psi and 90 kHz using SPHERO™ Rainbow Calibration Particles (RCP-30-5A).*
*MESF, can vary lot-to-lot

  • FITC : 87 molecules of equivalent soluble fluorochrome (MESF-FITC)
  • PE: 29 molecules of equivalent soluble fluorochrome (MESF-PE)
Fluorescence resolution
Coefficient of variation (CV)

  • PI: Area, <3.0%, full G0/G1 peak for PI-stained chicken erythrocyte nuclei (CEN)
  • Hoechst: Area, <3.5%, full G0/G1 peak for Hoechst-stained chicken erythrocyte nuclei (CEN)
Fluorescence linearity
Doublet/singlet ratio CEN stained with PI: 1.95–2.05 (488-nm laser) or Hoechst: 1.95–2.05 (405-nm laser)
Forward and Side Scatter Sensitivity
Sensitivity enables separation of fixed platelets from noise, identification of bacteria, and detection of 0.5-µm beads
Forward and Side Scatter Resolution
Scatter performance is optimized for resolving lymphocytes, monocytes, and granulocytes
Soft Performance - Drop Drive Frequency
Range: 1–100,000 Hz
Soft Performance - Purity and Yield
At 70 psi and 87 kHz with an average threshold rate of 25,000 events per second, a four-way sort achieved a purity of >98% and a yield >80% of Poisson’s expected yield. Higher threshold rates up to 70,000 events per second can be achieved without affecting purity. However, yield will decrease based on Poisson’s statistics
Soft Performance - Functionality
Dendritic cells (myeloid and plasmacytoid, mDs and pDCs, respectively) were isolated from the peripheral blood mononuclear cells of three donors and sorted on the BD FACSAria III system (one sort per donor) which uses the same cuvettebased flow cell design as the BD FACSAria Fusion. Post-sort cell viability was assessed using a
live/dead exclusion marker, and functionality was assessed by intracellular cytokine staining after 6 or 18 hours of stimulation with the TLR 7 & 8 agonist R848. Post-sort viability at 6–18 hours was >90% for all three donors. Both pDCs and mDCs for all three donors produced IFN-a, TNF-a, and IL-12 stimulation, demonstrating post-sort functionality
Soft Performance - Sort Collection Devices
  • Two-way sorting: 12 x 75-mm and 15-mL tubes
  • Four-way sorting: 1.5-mL microtubes and 12 x 75-mm tubes
  • Single cell sort into multiwall plates: 6-; 24-; 96-; 384-well plates
Soft Performance - FACS™ Accudrop
  • Red diode laser provided for fully automated drop-delay determination
  • Automated drop breakoff monitoring
  • Automated clog detection and sort tube protection system using Sweet Spot technology
Signal Processing -
Converter
10-MHz analog-to-digital converter. Pulse sampling is precisely matched to the particle flow rate in the cuvette. Particles travel slower compared to conventional stream-in-air sorters. This increases the light collected, resulting in better sensitivity. High-speed sorting is achieved by accelerating the stream through the nozzle, achieving drop rates comparable to stream-in-air sorters. The flow cell design and electronics are matched to maximize signal while maintaining maximum sort speed, purity, and yield
Signal Processing -
Workstation Resolution
262,144 channels
Signal Processing -
Pulse Processing
Height, area, and width measurements available for any parameter. Ratio measurements are also available
Signal Processing -
Time
Can be correlated to any parameter for kinetic experiments or other applications
  • Benchtop spectral flow cytometer
  • 5 spatially separated lasers
  • 64 fluorescent detectors
    • Sample loading options:
      • 5ml tubes
      • 96 – well plates

Description

The Cytek Aurora cytometer is a benchtop spectral flow cytometer. Equipped with 5 spatially separated lasers, 64 fluorescent detectors and 3 light scatter detectors for fast multicolour analysis with accurate data acquisition rate at up to 30,000 events per second (with beads). The Cytek Aurora cytometer is equipped with plate loader system for acquiring samples from a 96 well plates. Plate loader has controlled plate stage temperature within the range 4-30° C.

Applications

Multicolour flow cytometry analysis, high autofluorescence and background substraction, high speed data aquisition.

Specification

Detector configuration [PDF]

Excitation Optics
Laser power

  • 355 nm: 20 mW
  • 405 nm: 100 mW
  • 488 nm: 50 mW
  • 561 nm: 50 mW
  • 640 nm: 80 mW

Flat Top Laser beam profile with narrow vertical beam height optimized for small particle detection.
Emission Optics
Forward Scatter Detector: High-performance semiconductor detector with 488 bandpass filter
Side Scatter Detector: Two high-performance semiconductor detector with 405 and 488 bandpass filters

Fluorescence Detectors and Filters:
The Cytek Aurora cytometer is equipped with Proprietary high sensitivity Coarse Wavelength Division Multiplexing (CWDM) 8-16 - channel semiconductor detector array per laser enabling more efficient spectrum capture for dyes emitting in the 400-900 nm range. No filter changes required for any fluorochrome excited by the 355nm, 405nm, 488nm, 561nm and 640nm lasers.
Fluidics
  • Sample Flow Rates - Low: 20 μL/min, Medium: 40 μL/min, High: 80 μL/min
  • Sample input format: Manual (12x75mm polystyrene and polypropylene tubes) and automated (96 well plate) sample input formats
  • Sample dead volume: 5μl for 12x75mm tube
  • Sample restoration mode: only for 96 well plate loader
Fluorescence sensitivity
  • FITC : <110 MEFL
  • PE: <35 MEFL
  • APC: <15 MEFL
  • Pacific Blue: <190 MEFL

*measurements based on an average from three systems and performed using SPHERO Rainbow
Calibration Particle (RCP 30-5A) based on its peak emission channel
Fluorescence linearity
FITC R2 ≥0.995 / PE R2 ≥0.995
Forward and Side Scatter Sensitivity
Enables separation of fixed platelets from noise
Forward and Side Scatter Resolution
Performance is optimized for resolving lymphocytes, monocytes, and granulocytes
Side Scatter Resolution
Capable of resolving 0.2μm beads from noise
Carryover
<0.1%
Data Acquisition Rate
35,000 events/s
Spectroflo Software
  • Live unmixing during acquisition
  • Developed specifically to streamline assay setup, data acquisition, and file export
  • Automated QC module
  • Auto-fluorescence extraction
  • Raw and Unmixed FCS 3.1 files
Signal Processing
Digital signal processing with automatic window gate adjustment. 22-bit 6.5 log decades. Threshold using any single parameter or combination of parameters.
Pulse Shape Parameters
Pulse Area and Height for every parameter. Width for scatter parameters and one fluorescence parameter for each laser.