Cytometry and Antibody Technology

Mass Cytometry

Work in progress

Mass Cytometry, also known as CyTOF (cytometry by time of flight), is similar to flow cytometry but uses heavy metals tagged antibodies instead of fluorophore tagged antibodies. The metals used for CyTOF are non-rare, non-biological, and non-radioactive. There are several pros to using metal tags:

  • Abundant tags of similar intensity
  • Discrete signals: minimal overlap
  • Signal Overlap is minimal and predictable (fewer controls to run)
  • No compensation required
  • Zero autofluorescence background

 

Helios, a CyTOF Mass cytometer

  • Inductively Coupled Plasma (ICP) Time-of-Flight (TOF) mass spectrometry discriminates metal-conjugated probes on a per-cell basis
  • 75-209 Dalton atomic analytical mass range (135 Channels)
  • Single cell suspension input
  • .fcs (FCS 3.0) and .txt data output

In terms of samples that can be run on the Helios, it can run any type of single cell suspension. However precautions should be taken to avoid heavy metal contamination so we advise contacting the core prior to banking samples to ensure that samples are handled appropriately.

There are currently 44 metals available to conjugate to antibodies, so the maximum number of markers in a single panel is 44. It should also be noted that CyTOF does not have the ability to discriminate cell size (FSC and SSC in flow cytometry), so all cells must have antibodies to define them.

  • The sample staining procedure is similar to that for conventional cytometry. Once the staining procedure is complete, all samples must be fixed prior to running on the Helios
  • During sample collection and sample staining extra precautions should be taken to minimize heavy metal contamination. Please contact the core to discuss the precautions.
  • The flow rate of the Helios is much slower than flow cytometry – generally between 300-500 events/second.
  • The core provides extensive assistance for all CyTOF projects. The Scientific Associate Director will oversee the project, assisting with panel design and analyzing antibody titration experiments. Our Helios operator is responsible for running samples and maintaining the instrumennt – users will drop off prepared samples for the Helios operator to run. For data analysis of samples, we have previously collaborated with the Bioinformatics Core.
  • CyTOF data is typically analyzed with algorithms, but any standard flow cytometry analysis software could be used.

To begin a CyTOF project, please contact the Scientific Associate Director Laura Johnston. If possible, please inform her of your plans prior to sample banking so that she can assist with proper sample handling procedures.

Mass Cytometry/CytOF (Helios) Resources