Rapid purification of billions of circulating CD19+ B cells directly from leukophoresis samples.

The study of the biology and function of B cells, or the dissection and in vitro creation of enormous recombinant antibody repertoires, requires the isolation of large numbers of pure CD19+ B cells. The StraightFrom® Leukopak CD19 MicroBead Kit was recently introduced as a fast and robust kit to isolate human CD19+ B cells. This uses paramagnetic microbeads conjugated to high-affinity anti-CD19 monoclonal antibodies to bind B cells in leukapheresis (Leukopak) samples. The overall purity of the isolated cells, together with the characterization of the different CD19+ subclasses, was assessed by flow cytometry using a recombinant (REAffinity) antibody panel, revealing that the method allowed the recovery of over 93% of CD19+ B cells without any pre-purification step. This enables the relatively straightforward purification of all the circulating CD19+ B cells in a single donor.

Evolutionary trends and functional anatomy of the human expanded autophagy network.

All eukaryotic cells utilize autophagy for protein and organelle turnover, thus assuring subcellular quality control, homeostasis, and survival. In order to address recent advances in identification of human autophagy associated genes, and to describe autophagy on a system-wide level, we established an autophagy-centered gene interaction network by merging various primary data sets and by retrieving respective interaction data. The resulting network ('AXAN') was analyzed with respect to subnetworks, e.g. the prime gene subnetwork (including the core machinery, signaling pathways and autophagy receptors) and the transcription subnetwork. To describe aspects of evolution within this network, we assessed the presence of protein orthologs across 99 eukaryotic model organisms. We visualized evolutionary trends for prime gene categories and evolutionary tracks for selected AXAN genes. This analysis confirms the eukaryotic origin of autophagy core genes while it points to a diverse evolutionary history of autophagy receptors. Next, we used module identification to describe the functional anatomy of the network at the level of pathway modules. In addition to obvious pathways (e.g., lysosomal degradation, insulin signaling) our data unveil the existence of context-related modules such as Rho GTPase signaling. Last, we used a tripartite, image-based RNAi - screen to test candidate genes predicted to play a role in regulation of autophagy. We verified the Rho GTPase, CDC42, as a novel regulator of autophagy-related signaling. This study emphasizes the applicability of system-wide approaches to gain novel insights into a complex biological process and to describe the human autophagy pathway at a hitherto unprecedented level of detail.

Vacuum-assisted cell loading enables shear-free mammalian microfluidic culture.

Microfluidic perfusion cultures for mammalian cells provide a novel means for probing single-cell behavior but require the management of culture parameters such as flow-induced shear stress. Methods to eliminate shear stress generally focus on capturing cells in regions with high resistance to fluid flow. Here, we present a novel trapping design to easily and reliably load a high density of cells into culture chambers that are extremely isolated from potentially damaging flow effects. We utilize a transient on-chip vacuum to remove air from the culture chambers and rapidly replace the volume with a liquid cell suspension. We demonstrate the ability of this simple and robust method to load and culture three commonly used cell lines. We show how the incorporation of an on-chip function generator can be used for dynamic stimulation of cells during long-term continuous perfusion culture.