Phospho-Flow Cytometry
Overview
Phospho-flow cytometry (phospho-flow) detects intracellular phosphorylation of signalling proteins in single cells by staining with phospho-specific antibodies after fixation and permeabilisation. It allows direct measurement of signalling pathway activation (e.g., ERK, p38 MAPK, STAT) in phenotypically defined cell subsets without the need for cell sorting. In B cell immunology, phospho-flow is used to compare TLR, BCR, and CD40 signalling responsiveness across B cell populations.
Key Points from Literature
- TLR7 responsiveness of DN2 and aNAV cells: B cells stimulated with R848 (TLR7/8 agonist) and stained for phospho-ERK (pERK) and phospho-p38 MAPK (pMAPKp38). DN2 and aNAV cells showed significantly enhanced pERK and pMAPKp38 responses compared with rNAV, SWM, DN1, and NAV cells. This TLR7 hyper-responsiveness is the functional hallmark of EF pathway cells and is mechanistically explained by low expression of negative TLR regulators TRAF5 and TNFAIP3 (see Jenks2018 - DN2 B Cells and EF Pathway in SLE, phospho-flow on sorted/gated populations).
- CD40L unresponsiveness of DN2: CD40L stimulation increased CD25 expression on rNAV and SWM cells but failed to activate DN2 cells. This result, assessed by surface marker upregulation rather than phospho-flow per se, complements the TLR7 phospho-flow data to establish the dual functional phenotype: TLR7-hyper-responsive + CD40L-unresponsive (see Jenks2018 - DN2 B Cells and EF Pathway in SLE).
Contradictions & Debates
None documented in current wiki sources.
Related Pages
Conventional Flow Cytometry, DN2 B Cell, Activated Naive B Cell, TLR7, TRAF5, Extrafollicular Response