Time of flight mass cytometry (CyTOF)

Cells from separate donors or conditions are stained separately with Cisplatin (Cat. No. 201064, Fluidigm) followed by barcoding using the Cell-ID 20-Plex Pd Barcoding Kit (Cat. No. 201060, Fluidigm).

Following barcoding, samples are combined into a single 5 mL polystyrene U-bottom tube and incubated in the surface marker antibody cocktail for 30 min at 4 °C

Cells are then fixed using 2% PFA, for 20mins.

For intracellular staining, cells are permeabilized by incubation with Triton X 0.1% for 5 min at room temperature, followed by incubation with intracellular antibody cocktail for 30 min at 4 °C.

Stained cells are incubated overnight with Cell-ID Intercalator (Cat. No. 201192A, Fluidigm).

The following morning, cells are washed in either Maxpar PBS (Cat. No. 201058, Fluidigm), Maxpar Cell Staining Buffer (Cat. No. 201068, Fluidigm), or Millipure Water, with a spin at 1600 RPM for 4 min to remove the supernatant and replace it with fresh buffer.

Samples are run on a time of flight mass cytometer (CyTOF 2, Fluidigm) and analyzed with Cytobank (Cytobank Inc., RRID:SCR_014043) and Astrolabe Diagnostics (https://astrolabediagnostics.com/)