The Human Immune Monitoring Center (HIMC)

Mass Cytometry

Mass cytometry, or CyTOF (Fluidigm), is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorochromes.  Readout is by time-of-flight mass spectrometry.  This allows for the combination of many more antibody specificities in a single samples, without significant spillover between channels.  We have standardized panels for  immunophenotyping, phenotyping+intracellular cytokine staining (ICS), and phenotyping+phospho-CyTOF, each with 30 to 38  antibodies.   Contact mleipold@stanford.edu for details.

Immunophenotyping:  Our 33-antibody CyTOF immunophenotyping panel is designed for use with cryopreserved PBMC, though it could be adapted for other sample types.  A limited number of additional antibodies can be added to open channels, subject to availability.

Intracellular cytokines:  Our 38-antibody CyTOF ICS panel is aimed primarily at dissection of T cell responses.  Limited substitutions are possible (with common ones as listed).

Phospho-CyTOF:  Our 30-antibody phospho-CyTOF panel can be used with the indicated stimuli to read out a broad array of signaling pathways in many different cell subsets.  Additions or substitutions in this panel are more difficult, due to the requirement for surface marker antibodies to work well on fixed epitopes.

See the list of standard antibodies in these three CyTOF panels here. (revised November 2023)

For a video interview with Dr. Mike Leipold on CyTOF experimental issues, see https://www.youtube.com/watch?v=UYYq9jh7FgQ.

User Instructions

1.  Planning your assay:  CyTOF assays are run in batches of up to 12 samples.  If you have more samples, please try to randomize your batches so that, for example, cases and controls are not segregated into separate batches.

2.  Collecting your samples:  Please use an optimized CRYOPRESERVATION PROTOCOL for viable PBMC.  We recommend to freeze 10^7 cells per cryovial, and supply one cryovial for each of the above assays.  PBMC processing and cryopreservation services are also available through our Blood Processing Unit.

3.  Running your samples:  An online order is required before samples can be run. Turnaround time varies depending on the size of your project and the current workload; smaller projects can usually be completed within 3-4 weeks of order placement and sample delivery.

4.  Data analysis:  We provide a standard data analysis using FlowJo software.  An Excel report with percentages of all gated populations, as well as pdf's of the gating results and all fcs files, are provided via Google Drive.  For standard panels, the Excel data is simulataneously uploaded to our online database, Stanford Data Miner, where it can be integrated with other HIMC data from your lab, as well as clinical/demographic data, if provided.  Click on the help link in the upper left corner of the Data Miner homepage for instructions on using this system.  

Statistical consultation is available through the HIMC Statistical Consulting Service.

Protocols

Please see our Protocols page for detailed lab protocols as well as short descriptions suitable for publications or grant applications.

Addtional information:

• Mass Cytometry Review (Trends in Immunology)
• Analyzing CyTOF data in FlowJo