Resources
LMA CyTOF Core Resources
Cytoforum
The Cytoforum is the best place to connect with the greater CyTOF community on a variety of topics ranging from experimental design, instrument operation, data analysis and more. Search older posts, ask your questions, get information on publications, and answer questions in your area of expertise.
Panel Design
An antibody panel design tool and supporting documents are available by creating an account with Fluidigm. Please be sure to read the Panel Design User Guide before utilizing the design tool.
Antibodies & Reagents
Pre-conjugated antibodies are available from Fluidigm and from the Antibody Core at Brigham and Women's Hospital (BWH). Fluidigm also provides pre-designed panel kits, sample prep reagents, barcoding kits. and Max-Par antibody conjugation kits to conjugate your own antibodies.
Antibodies for conjugation MUST be carrier-free. Biolegend, Miltenyi and Cell Signaling Technology offer Maxpar Ready and CyTOF validated antibodies. Other companies will require special requests to purchase antibodies carrier-free.
Cell Signaling CyTOF and Maxpar Ready
Protocols
Protocols for antibody conjugation, cell surface, viability, cytoplasmic targets, nuclear targets, signaling proteins and phosphoproteins can be found on Fluidigm's website by scrolling down to the Protocol section, under Resources. They can also be accessed when logged into the panel design website.
The Nolan Lab at Stanford has protocols available for stimulation, cell surface, methanol permeabilization and saponin permeabilization. They also provide protocols for validating and titrating antibodies, Maxpar antibody conjugation, and removal of carrier buffers in antibodies.
eBioscience's FoxP3 kit is popular for all panels using FoxP3. To use the kit:
- Preform viability staining with 195Pt or 103Rh according to the protocol included with the reagent.
- Follow the eBioscience FoxP3 kit protocol as described.
- Use the Fluidigm protocol for Ir191/193 intercalator staining in Fluidigm Fix/Perm buffer (adding an additional 4% PFA to the buffer).
- After staining do at least 2 washes in cell staining buffer and 2 washes in water, then re-suspend in 1:10 EQ Beads prior to running on CyTOF.
Data Analysis Tools and Information
Commercially available software can be used to analyze your CyTOF data. Cytobank, FlowJo and FCS Express can be used for basic analysis such as bivariate plots, histogram overlays, heatmaps and dose response. Cytobank and FlowJo have plugins, which offer user-friendly versions of clustering algorithms such as ViSNE/tSNE, SPADE, and Citrus for analyzing high dimensional data. Maxpar Pathsetter can be used on MDIPA-stained samples to automatically analyze and identify 37 immune cell types.
Free software designed for MatLab and R can be downloaded from the Nolan Lab. They offer overviews and instructions for each type of analysis available. ACCENSE, an alternative tSNE algorithm, is also available for download independently. Premessa is an R script that can be used to edit/rename panels as well as normalize and debarcode FCS files.