The various ADAMTS13-specific monoclonal antibodies described by Soejima and coworkers (15
) (Wh2-11-1, Wh2-22-1A, Wh-10, W688X6-1 and W688X3-69) and the commercially available ADAMTS13 polyclonal antibody BL154G were previously tested to detect ADAMTS13 on Western blots (http://www.natutec.de/pdf_bethyl/A300-391A.pdf
). Here we used flow cytometry to test these antibodies for their efficacy in detecting the endogenous ADAMTS13 in HEK293 cells (). A preliminary test of both isotype controls alongside a secondary-only control was conducted (Figure S1
) revealing comparable histogram curves and median signal measurements. All figures contain histograms of secondary-only data to ensure clarity, although all three controls were conducted for each experimental setup. Significantly high fluorescence intensities were measured for each antibody relative to control staining with secondary antibody only or anti-mouse IgG2aκ or IgG1κ isotypic antibodies (). Of the antibodies that we tested, Wh2-11-1 had the highest reactivity to ADAMTS13. Therefore, we used this antibody alongside the polyclonal antibody BL154G to study the endogenous expression of ADAMTS13 in multiple liver cell lines: 7404, Alexander, Hep3b, Huh-7 and LX2 (human hepatic stellate cells). In addition, the cells were fixed and permeabilized with two alternative methods as discussed in the Materials and Methods
section, or left untreated (unfixed and unpermeabilized) prior to incubating with the ADAMTS13 antibodies. Following treatment with the primary antibody, the cells were stained with the secondary antibody and analyzed by flow cytometry. These results are depicted in and clearly demonstrate that histogram shifts occur using both ADAMTS13 antibodies; these shifts are dramatically pronounced when the cells are permeabilized. Finally, among all the cell lines tested, LX2, the liver stellate cell line (19
), showed the highest median fluorescence (). This observation is consistent with the report of Fujimura and coworkers that liver stellate cells show the highest expression of ADAMTS13 and are the major contributors of ADAMTS13 found in blood (4
). The median fluorescence of the permeabilized cells in this assay is a measure of relative ADAMTS13 protein. Endogenous levels of intracellular ADAMTS13 were not detected using conventional cell lysate preparation and Western blotting procedures (data not shown). This finding adds to the significance of detecting endogenous levels of intracellular ADAMTS13 via flow cytometry.
Flow cytometry detection of intracellular ADAMTS13 in HEK293 cells using various specific anti-ADAMTS13 monoclonal antibodies
Detection of ADAMTS13 intracellular expression in various liver ADAMTS13-expressing cell lines
As a further demonstration of this method’s sensitivity, transient expression of ADAMTS13 was knocked down in HEK293 cells using siRNA as described in Materials and Methods. Upon targeted siRNA transfection, median fluorescence fell to 31.91/41.05 a.u. from 55.73/69.78 a.u for Wh2-11-1 and anti-V5 respectively (). With isotypic readings subtracted, cells probed with Wh2-11-1 showed a 68% reduction in expression, while anti-V5 probed cells showed a 60% decline. One reason for this slight difference may be that the use of Wh2-11-2 estimates the reduction in both endogenous and transfected ADAMTS13 while the use of the anti-V5 antibody only estimates the reduction in the transfected ADAMTS13. Conversely, cells transfected with control scrambled siRNA retained 73% and 80% of their expression, when probing with Wh2-11-1 and anti-V5 respectively. A similar reduction in ADAMTS13 expression was observed through Western blotting. Relative to cells transfected with plasmid ADAMTS13 alone, cells administered scrambled siRNA retained 79% of ADAMTS13 expression, while targeted siRNA transfection obliterated detectable levels of ADAMTS13 ().
Measurement of transient ADAMTS13 expression via FACS after siRNA knock-down
Analogously, we used Wh2-11-1 and an antibody against the C-terminal V5 tag of recombinant ADAMTS13 to detect gains in ADAMTS13-specific antibody reactivity which is reflected by gains in median fluorescence upon transfection with plasmid ADAMTS13 (pADAMTS13) in HEK293 cells. The anti-V5 antibody detects the recombinant protein only, while Wh2-11-1 measures total ADAMTS13 within the cell. Both antibodies yielded a significant gain in detection following transfection with pADAMTS13 (). The median fluorescence observed was always higher in pADAMTS13-transfected cells than in cells transfected with the empty vector control, which was verified by an immunoblot of cell lysates using the same two antibodies (). To accompany this flow cytometry study, confocal imaging of ADAMTS13 following pADAMTS13 transfection was performed. The increase in intracellular expression of ADAMTS13 following transfection is clearly evident in images of immunolabeled HEK293 cells using the same antibodies—anti-V5 and Wh2-11-1 ().
Finally, dose response experiments were conducted to determine the sensitivity of flow cytometry to various amounts of transfected DNA and transfection regent used during the process of transfecting pADAMTS13 into HEK293 cells. The response to increasing concentrations of DNA is clearly reflected in the increasing median fluorescence intensity measured by flow cytometry using Wh2-11-1 (). The saturation point was reached when using 5 µg DNA per 5 × 105 cells in the transfection procedure. In another experiment, we varied the amount of Fugene6 transfection reagent but retained a constant amount of DNA (2 µg) (). In this experiment, we found a progressive increase in the percentage of transfected cells as we increased the concentration of the transfection reagent. With the use of 3, 6, 8 and 12 µL of Fugene6 transfection reagent, the percentage of transfected cells increased to 2.0, 5.3, 35.3 and 37.8, respectively. A further increase in the quantity of transfection reagent yielded no further increase in the transfected cells, and the percent of stained cells reached a plateau. The nature of this incremental increase in fluorescence signal obtained by flow cytometry clearly shows a dose response curve vis-à-vis the amount of DNA used to transfect the cells as well as the transfection reagent itself.
Detection of intracellular ADAMTS13 in transfected HEK293 via flow cytometry, Western blotting and confocal microscopy
ADAMTS13 is an unusual secreted protein, as it has both intracellular and extracellular functionality (8
). We measured the activity of the secreted protein using the fluorescent substrate FRETS-VWF73, as described previously by Kokame and coworkers (20
). As expected, an increase in intracellular ADAMTS13 measured by flow cytometry accurately predicts increased proteolytic activity of extracellular ADAMTS13 (data not shown), due to higher secretion levels of ADAMTS13 into the culture media following transfection.
Here, we have introduced flow cytometry as a novel method to detect and quantitatively measure expression levels of intracellular ADAMTS13. We have shown that the expression level of intracellular ADAMTS13 as determined by flow cytometry correlates well with previously established assays for ADAMTS13 detection: Western blotting, confocal imaging and FRETS-VWF activity assay.
The method described here, unlike immunoblotting of cell lysates, easily lends itself to high throughput assays. Thus, the technique could be useful in the standardization of protocols for the production of recombinant ADAMTS13. In addition, during large-scale production of ADAMTS13 this method would be useful in monitoring the expression levels at frequent intervals to ensure the quality of the protein, which is the most crucial issue for the production of any recombinant therapeutic protein.