Institute for Immunity, Transplantation and Infection

The Human Immune Monitoring Center (HIMC)

Mass Cytometry

For more information

Contact:

Mike Leipold, CyTOF Manager
Email » [mleipold]

Address:
Human Immune Monitoring Center
Fairchild Science Bldg, D033
299 Campus Drive
Stanford, CA 94305-5124

phone:  650-723-4984
fax:  650-498-7495

Mass cytometry, or CyTOF (DVS Sciences), 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 an immunophenotyping CyTOF panel with 23 antibodies, with other panels to come shortly.  Experienced CyTOF users can also schedule time on the instrument to run independent experiments; contact mleipold@stanford.edu [mleipold] for details.

Immunophenotyping:  Our 23-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.

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 provided 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, are posted to our Basecamp project management system.  New users will recieve an email invitation to join this system.  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.  Further analysis, such as can be done using the SPADE clustering algorithm, is available for an additional fee.  Click here for instructions on analyzing CyTOF data in FlowJo.

Protocols

1.  Short protocol descriptions, suitable for publications or grant applications:

CyTOF Immunophenotyping:  This assay was performed in the Human Immune Monitoring Center at Stanford University.  PBMCs were thawed in warm media, washed twice, resuspended in CyFACS buffer (PBS supplemented with 2% BSA, 2 mM EDTA, and 0.1% soium azide), and viable cells were counted by Vicell. Cells were added to a V-bottom microtiter plate at 1.5 million viable cells/well and washed once by pelleting and resuspension in fresh CyFACS buffer. The cells were stained for 60 min on ice with 50 uL of the following antibody-polymer conjugate cocktail: [insert Ab list here]. All antibodies were from purified unconjugated, carrier-protein-free stocks from BD Biosciences, Biolegend, or R&D Systems. The polymer and metal isotopes were from DVS Sciences. The cells were washed twice by pelleting and resuspension with 250 uL FACS buffer. The cells were resuspended in 100 uL PBS buffer containing 2 ug/mL Live-Dead (DOTA-maleimide (Macrocyclics) containing natural-abundance indium). The cells were washed twice by pelleting and resuspension with 250 uL PBS. The cells were resuspended in 100 uL 2% PFA in PBS and placed at 4C overnight. The next day, the cells were pelleted and washed by resuspension in fresh PBS. The cells were resuspended in 100 uL eBiosciences permeabilization buffer (1x in PBS) and placed on ice for 45 min before washing twice with 250 uL PBS. If intracellular staining was performed, the cells were resuspended in 50 uL antibody cocktail in CyFACS for 1 hour on ice before washing twice in CyFACS. The cells were resuspended in 100 uL iridium-containing DNA intercalator (1:2000 dilution in PBS; DVS Sciences) and incubated at room temperature for 20 min. The cells were washed twice in 250 uL MilliQ water. The cells were diluted in a total volume of 700 uL in MilliQ water before injection into the CyTOF (DVS Sciences). Data analysis was performed using FlowJo v9.3 (CyTOF settings) by gating on intact cells based on the iridium isotopes from the intercalator, then on singlets by Ir191 vs cell length, then on live cells (Indium-LiveDead minus population), followed by cell subset-specific gating.

2.  Detailed protocols and addtional information:

Stanford Medicine Resources:

Footer Links: