ABSTRACT
Conventional cell trapping methods using microwells with small dimensions (10-20 µm) are useful for examining the instantaneous cell response to reagents; however, such wells have insufficient space for longer duration screening tests that require observation of cell attachment and division. Here we describe a flow method that enables single cell trapping in microwells with dimensions of 50 µm, a size sufficient to allow attachment and division of captured cells. Among various geometries tested, triangular microwells were found to be most efficient for single cell trapping while providing ample space for cells to grow and spread. An important trapping mechanism is the formation of fluid streamlines inside, rather than over, the microwells. A strong flow recirculation occurs in the triangular microwell so that it efficiently catches cells. Once a cell is captured, the cell presence in the microwell changes the flow pattern, thereby preventing trapping of other cells. About 62% of microwells were filled with single cells after a 20 min loading procedure. Human prostate cancer cells (PC3) were used for validation of our system.
ABSTRACT
We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000-g fraction to the 200,000-g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000-g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000-g fractions. LC-MS/MS analysis showed that markers of "intracellular vesicles," chiefly endosomal markers, were present in both the 17,000- and the 200,000-g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000-g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000-g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000-g fractions are highly heterogeneous and cannot be equated with "plasma membrane" and "intracellular vesicle" fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.
