Sep 01, 2022
Time of flight mass cytometry (CyTOF)
- 1University of Minnesota
External link: https://doi.org/10.3389/fimmu.2023.1060905
Protocol Citation: Peter Hinderlie, Philippa R Kennedy 2022. Time of flight mass cytometry (CyTOF). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo522dl4o/v1
Manuscript citation:
Kennedy PR, Vallera DA, Ettestad B, Hallstrom C, Kodal B, Todhunter DA, Bendzick L, Hinderlie P, Walker JT, Pulkrabek B, Pastan I, Kratzke RA, Fujioka N, Miller JS, Felices M, A tri-specific killer engager against mesothelin targets NK cells towards lung cancer. Frontiers in Immunology doi: 10.3389/fimmu.2023.1060905
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 22, 2020
Last Modified: September 01, 2022
Protocol Integer ID: 36064
Abstract
Staining procedure for cells analyzed by time of flight mass cytometry.
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/)