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.