High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry.

Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations.

Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling.

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca(2+)) influx occurs via highly selective Ca(2+) release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca(2+)-dependent signaling events and formation of the immune synapse.