A radial calibration window for analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments.