Detailed staining protocols have been described (42 (link), 43 (link)). Briefly, cells were transferred to 96-well deep-well plates (Sigma), resuspended in 25 μM cisplatin (Enzo Life Sciences) for 1 min (44 (link)), and quenched with 100% serum. Cells were stained for 30 min on ice, fixed (BD FACS Lyse), permeabilized (BD FACS Perm II), and stained with intracellular antibodies for 45 min on ice. Staining panels are described in table S1 . All antibodies were conjugated using MaxPar X8 labeling kits (DVS Sciences). Cells were suspended overnight in iridium interchelator (DVS Sciences) in 2% paraformaldehyde in phosphate-buffered saline (PBS) and washed 1× in PBS and 2× in H2O immediately before acquisition. Stained cells were analyzed on a mass cytometer (Fluidigm).
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Mass Cytometry Staining Protocol
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Corresponding Organization : Stanford University
Other organizations : Yale University, University of Washington, Kenyatta National Hospital
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Variable analysis
independent variables
- Cisplatin concentration (25 μM)
- Cell staining with intracellular antibodies
- Cell transfer to 96-well deep-well plates
- Cell resuspension in 25 μM cisplatin for 1 min
- Quenching with 100% serum
- Cell staining for 30 min on ice
- Cell fixation (BD FACS Lyse)
- Cell permeabilization (BD FACS Perm II)
- Intracellular antibody staining for 45 min on ice
- Cell suspension in iridium interchelator in 2% paraformaldehyde in PBS
- Cell washing in PBS and H2O before acquisition
- Positive controls not explicitly mentioned
- Negative controls not explicitly mentioned
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