High-parameter T-cell spectral phospho-flow-cytometry

Phospho-flow is an invaluable tool for investigation into immunological signaling, enabling concurrent staining of cell lineage markers alongside sensitive intracellular phospho-proteins. Phospho-flow holds promise as a powerful diagnostic and therapeutic tool that can enable signal profiling, cell phenotyping, drug screening, pharmacodynamic profiling and assessment of drug efficacy across multiple cell types. When combining phospho-flow with fluorescent cell barcoding (FCB) multiplexing technique, high throughput flow cytometry can be achieved. FCB enhances experiment robustness while reducing variability in staining and decreasing antibody usage. Despite its utility, inter-operator technique variability persists, highlighting the need for protocol and analysis standardization. Here we describe an experimental mechanism for stimulating mouse and human T cells that demonstrates robust activation that can be adapted for various experimental designs.