Cell Sorting Instrumentation

Our BSL2+ room C315 houses the two BD FACSAria cell sorter instruments.

The BD FACSAria I was upgraded in 2010 and comes equipped with 12 fluorescent PMTs over four lasers (405 nm violet, 488 nm blue, 561 nm yellow-green, and 640 nm red). This configuration is similar to the LSRII B analyzer, but with less options off the violet laser. The instrument operates with a 100 micron nozzle/20 PSI or 70 micron nozzle/35 PSI configuration. Detailed specifications can be found in the menu on the right. The Aria I is placed in a Bio-Bubble negative-pressure biosafety cabinet that provides the BSL2+ environment to protect the operator.

The BD FACSAria II is equipped with four lasers (355 nm UV, 405 nm violet, 488 nm blue, and 633 nm red) and has 13 fluorescent detectors. This configuration is similar to the Fortessa B analyzer, but with less options off the violet laser. Current options for configuration on the Aria II are 100/20; 85/45; 85/30; 70/35, and 130/10 (nozzle size/PSI). Detailed specifications can be found in the menu on the right.

Both instruments have the capacity to do one- to four-way sorts into a variety of tube styles, such as 12x75 mm tubes of any material, Eppendorfs, PCR tubes, and 15-ml conical tubes (2-way only). They can also sort into plates (6, 12, 24, 48, 96, and 384 wells) and onto slides.

Our cell sorters undergo a stringent routine maintenance and cleaning protocol to warrant aseptic sorting conditions at all times. We use sterile-filtered HEPES-buffered saline with EDTA as sheath fluid.

For magnetic cell sorting, our facility houses a Miltenyi Classic AutoMACS. This instrument is located in a biosafety cabinet in Rm C322, and does not require operator assistance. Columns, clean bottles, and cleaning solution are provided. The user is responsible for running buffer and beads. Daily usage charges apply. Training is available through Kitty de Jong x8416.

Technical Specifications

  • Quartz cuvette flow cell is gel-coupled by refractive index-matching optical gel to the fluorescent objective lens. Sort fractions collected jet-air
  • 4 Laser - separate interrogation of sample for each laser
  • 14 parameters: 12 fluorescent and 2 scatter parameters + Time
  • Sheath pressure is adjustable from 2 to 75 psi
  • At 70 psi and 90 kHz with an average threshold rate of 25,000 events per second for a four-way sort results in a purity of > 98% and a yield > 80% of Poisson's expected yield
  • Sample Collection Cooling 4°, 20°, 37°, and 42°C (Water recirculator for refrigeration/heating)
  • Aerosol Management Option (AMO) with Bio-Bubble negative pressure. Bio Safety Level 2+ environment
  • 262,144 channel (18 bit) dynamic range on all parameters, Windows XP-Pro OS, Diva software acquisition

Excitation Sources

  • 405nm, Point Source Violet solid state: 30 mW
  • 488nm, Coherent® Sapphire™ solid state: 20mw
  • 561nm, Coherent solid state: 35mW
  • 633nm, HeNe air-cooled: 20mW

Emission Detected

  • 405nm Violet Laser intercept:
    • 450/50nm Pac Blue, BV421
    • 530/30nm AmCyan BV510
  • 488nm Blue Laser intercept:
    • 488/10nm FSC
    • 488/10nm SSC
    • 530/30nm FITC/Alexa 488
    • 710/50nm PerCP, PerCP-Cy5.5
  • 561nm (Yellow/ Green) intercept:
    • 585/15nm PE, dsRed
    • 610/20nm PE-TxRd/mCherry
    • 670/30nm PE-Cy5
    • 710/50nm PE-Cy5.5
    • 780/60nm PE Cy7
  • 633nm Red Laser intercept:
    • 660/20nm APC
    • 730/30nm Alexa-700
    • 780/60nm APC-Cy7

 

Technical Specifications

  • Quartz cuvette flow cell is gel-coupled by refractive index-matching optical gel to the fluorescent objective lens. Sort fractions collected jet-air
  • 4 laser
  • 15 parameters: 13 fluorescent and two scatter parameters + Time
  • Sheath pressure is adjustable from 2 to 75 psi.
  • At 70 psi and 90 kHz with an average threshold rate of 25,000 events per second for a four-way sort results in a purity of > 98% and a yield > 80% of Poisson's expected yield
  • Aerosol Management Option (AMO)
  • 262,144 channel (18 bit) dynamic range on all parameters
  • Windows XP-Pro OS, Diva software acquisition

Excitation Sources

  • 355nm Uniphase Xcyte 100nW
  • 405nm Coherent cube variable power thru software 100mW
  • 488nm Coherent® Sapphire™ solid state variable power thru software: 50-150mW
  • 640nm Coherent Cube variable power thru software 40mW

Emission Detected

  • 488nm laser interept:
    • 488/10 FSC
    • 488/10 SSC
    • 530/30nm FITC
    • 575/25nm PE
    • 610/20nm PE-Texas Red™®
    • 710/50nm PerCP -Cy5.5, PE-Cy5.55
    • 780/60nm PE-Cy™7
  • 640nm laser interept:
    • 660/20nm APC
    • 730/30nm Alexa-700
    • 780/60nm APC-Cy7
  • 405nm laser interept:
    • 450/50nm DAPI, Pacific Blue, BV421
    • 530/30nm AmCyan, BV510
    • 650nmLP Side Population
  • 355nm laser interept:
    • 379/28nm BUV395
    • 460/14nm DAPI, Hoechst

 

  • The autoMACS™ Separator is a benchtop instrument for high-speed automated cell sorting. Employing the MACS® Technology, the autoMACS Separator is designed for positive selection as well as depletion of magnetically labeled cells. The autoMACS Separator is operated with pre-set separation programs, thus, allowing optimization of cell sorting approaches according to cell abundance and the intensity of marker expression. The separated cells are immediately ready for experiments, cell analysis, or further subset sorting.
    • Fast—up to 4×109 magnetically labeled cells are sorted automatically within a few
      minutes.
    • Efficient—rare cells can be enriched up to 10,000-fold or unwanted cells may be depleted up to 99%.

MACS® Cell Separation Technology is available for human, mouse, rat and non-human primate cell isolation. For any other cell type MicroBeads for indirect magnetic labeling can be used.

  • Principle of the technology: MACS Cell Separation is based on MACS MicroBeads, specific monoclonal antibodies conjugated to superparamagnetic 50 nm particles. Labeled cells are retained in the magnetic field within a MACS Column. Separated cells—the positively labeled fraction as well as the non-labeled fraction—can directly be used for downstream applications, such as cell analysis, further expansion, or functional assays. The separated cells remain viable and their functionality is not compromised. 
  • Common applications: positive and negative separation of specific immunophenotypes. Enrichment of target cells before flow cytometric cell sorting to improve sorting efficiency. For a full repertoire of available analytes please see the Miltenyi Biotec website: www.miltenyibiotec.com.
  • AutoMACS Applications contacts:
    • Kitty de Jong: extension 8416
    • Hans Minderman: extension 4163
    • Paul Wallace: extension 4163