The generation of antibody-secreting cells (ASCs) is mainly regulated by follicular T helper cells (T FH) within germinal centers (GCs) and is crucial for the development of high-affinity antibodies.
One of the cornerstones of the adaptive immune system is the ability of B cells to respond to pathogens and differentiate into antibody-secreting cells. This will aid the further investigation of B cell responses in both health and disease. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation.
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