Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound bio-molecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 bio-sensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.
Mitochondria experience continuous fusion and fission in a living cell, but their dynamics remains poorly quantified. Here a theoretical model was developed, upon a simplified population balance equation (PBE), to predict the morphological changes induced by mitochondrial fission and fusion. Assuming that both fission and fusion events are statistically independent, the survival probability of mitochondria staying in the fission or fusion state was formulated as an exponentially-decayed function with time, which depended on the time-dependent distribution of the mitochondrial volume and the fission and fusion rates. Parametric analysis was done for two typical volume distributions. One was Gamma distribution and the other was Gaussian distribution, derived from the measurements of volume distribution for individual mitochondria in a living cell and purified mitochondria in vitro. The predictions indicated that the survival probability strongly depended on morphological changes of individual mitochondria and was inversely correlated to the fission and fusion rates. This work provided a new insight into quantifying the mitochondrial dynamics via monitoring the evolution of the mitochondrial volume.
WANG ShiQi 1,2,4 , FU ChangLiang 1,2 , ZHANG Yan 1,2 , CHEN Quan 3 & LONG Mian 1,2 1 Key Laboratory of Microgravity (National Microgravity Laboratory), Beijing 100190, China
Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts.The underlying mechanism,however,of cellular sensing and responding to the gravity vector is poorly understood.This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-,downward-or edge-on-oriented substrates.Cell morphology and nuclear translocation,cell proliferation and the cell cycle,and cytoskeletal reorganization were found to vary significantly in the three orientations.All of the responses were duration-dependent.These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.
LI HongCHEN JuanZHANG YanSUN ShuJinTAO ZuLai&LONG Mian
L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into under-standing how L-selectin shedding regulates leukocyte rolling and adhesion.
JIA XiaoLing1,2,3, CHEN Juan1,2 & LONG Mian1,2 1 National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100190, China
Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microflu- idic-based assay, however, remains to be further tested. In the current work, we developed a microflu- idic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quan- tified by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent ELISA-based cell binding assay. These results demonstrated that the system developed provided a new platform to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses.
YANG Fan, GAO YuXin, ZHANG Yan, CHEN Juan & LONG Mian National Microgravity Laboratory and Center for Biomechanics and Bioengineering, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100080, China
