Confocal microscopy

Cells were viewed using a spinning disk confocal microscope (Perkin Elmer) with a Volocity acquisition system, built on a Zeiss inverted microscope. Cells were imaged as soon as possible, normally one or two days after staining, a maximum of two weeks after staining. Cover glasses were cleaned with ethanol before they were viewed.

Images were captured using either a x63 or x100 immersion oil objective. Representative images of the cells were taken from two to three slides for each experiment to minimise the amount of bleaching taking place. Images were taken from a number of different fields in each slide, well separated from each other again to minimise bleaching. The confocal microscope takes images at different levels within a specimen producing ‘optical’ sections. This sequence of ‘optical sections’ is

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known as a z series. The z series is collected by coordinating the movement of the fine focus of the microscope with image collection. When images of a desired cell (or group of cells) within a field of view were to be taken the microscope was set to image from the region just above the cells down to the region just below the cells. This ensured that only the images required were taken (i.e. so that images of areas devoid of cells were not taken). This was to prevent bleaching, exposing the cells to as little light as possible.

The spacing between each image in a z series (the z-space) was set to 0.359μm for each x63 and x100 objective. This spacing fulfilled the Nyquist criteria. According to the Nyquist sampling theorem (Oppenheim et al, 1983) in order to resolve two points and to avoid under or over-sampling, the z-spacing should be equal to the axial resolution between the two points divided by at least two. Voxels (the three dimensional equivalent of pixels), larger than those specified by the Nyquist criteria would under sample an image, create artefacts and result in false colocalisation. Collecting images with voxels smaller than the Nyquist value would result in the re- imaging of sections and longer exposure times which could lead to photobleaching.

Untransfected, unlabelled cells were imaged to assess whether autofluorescence was produced by the cells. There was no apparent autofluorescence. Single labelled controls (i.e. FITC or Cy3 staining alone) were used to assess bleed-through. Bleed- through is the passage of fluorescence emission in an inappropriate detection channel caused by an overlap of emission spectra (Bolte & Cordelieres, 2006). There was no apparent bleed-through.

Cross-talk occurs when several fluorochromes are excited with the same wavelength at a time because their excitation spectra partially overlap (Bolte & Cordelieres, 2006). Cross talk was minimised by sequentially scanning the sample: one laser was activated and the corresponding emission collected, followed by excitation of the next laser and detection of emission.

For each experiment the exposure times were first set for each channel to be imaged using a randomly chosen cell expressing WT ADAMTS13. The same exposure times were then used to image negative control samples. Test samples (WT and mutant

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protein) were then imaged using the same exposure times. At each position images were taken in the order FITC, Cy3 and then DAPI. The objective then moved to the next position to take the next image. This method of imaging the cells as opposed to imaging all the FITC staining of each z stack followed by imaging all the Cy3 staining is more suitable for colocalisation analysis as it prevents the shifting of voxels between images.

To view DAPI staining, excitation was at 405nm and emission at 455/460nm. To view FITC staining, excitation was at 488nm and emission at 527/555nm. Finally for Cy3 staining excitation was at 568nm and emission at 615/670nm.

2.9 Immunofluorescence quantitation

The degree of colocolisation between ADAMTS13 and the Golgi was quantified using Volocity 3D image analysis software (Perkin Elmer). The ‘co-localization’ option was selected under the quantitation tab.

The images obtained after confocal microscopy were represented as a series of voxels, akin to pixels but representing space in addition to length and width. These voxels were given a value for each channel (wavelength) in which images were taken. A 14 bit channel was used to image the cells so each voxel was given a value between 0 and 16384 depending upon the intensity of the signal captured by the microscope, in this case having three different values for the three different wavelengths at which the images were viewed. For colocalisation analysis channel 1 was set to show the values obtained at a wavelength of 568nm. At this wavelength the fluorescently labelled Golgi was measured. Channel 2 was set to show the values obtained at a wavelength of 488nm. At this wavelength the fluorescently labelled ADAMTS13 could be measured.

Thresholds were first set to exclude voxels from the analysis which constituted background ‘noise.’ These were set by selecting a region of the image in which there were no cells. The software then calculated the threshold value for each channel separately by calculating the mean value of the voxel intensities (in a particular

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channel) in the region of interest plus three standard deviations (SD). The same threshold for each channel was used throughout the analysis.

Clearly defined cells within an image were selected for analysis. The region of interest was selected using the freehand tool. Only cells with no saturated voxels were used to carry out the quantitation analysis. If there were other cells or parts of cells within the same region of the Z-stack, in the region of interest, these cells were excluded from the analysis.

2.10 Proteasome/lysosome inhibition

Confluent HEK 293 cells plated in 90mm petri dishes (VWR), stably expressing ADAMTS13 were incubated with either 10M MG132 (carbobenzoxy-Leu-Leu- leucinal) (Merck Chemicals), 6M ALLN (N-acetyl-L-leucinyl-L-leucinyl- norleucinal) (Merck chemicals, prepared in DMSO according to the manufacturer’s instructions), 0.1µM Bafilomycin A1 (Sigma, prepared in DMSO according to the manufacturer’s instructions), 50mM ammonium chloride (prepared in sterile water) or with 0.1% DMSO (Sigma). After 5 hours cell lysate and supernatant samples were harvested as described (Chapter 2.3.2). Supernatant was concentrated as described above ~100 fold (Chapter 2.3.4). For SDS gel electrophoresis, 15µl of cell lysate and 30µl of supernatant was loaded.

2.11 Betaine experiments

Betaine is a chemical chaperone and has been recently shown to aid the secretion of a Factor VIII mutant (Roth et al, 2012). In order to investigate whether betaine could aid the secretion of an ADAMTS13 secretion defect mutant, confluent HEK 293 cells plated in 90mm petri dishes (VWR) stably expressing ADAMTS13 were incubated in the presence or absence of 100mM betaine (prepared in sterile water). After 96 hours cell lysate and supernatant samples were harvested as described previously (Chapter 2.3.2). Supernatant was concentrated as described previously ~100 fold (Chapter 2.3.4).

78 2.11.1 SDS-PAGE

For SDS-PAGE (polyacrylamide gel electrophoresis), 15µl of cell lysate and 30µl of supernatant was loaded. To ensure better separation of cell lysate samples, samples were also run on Criterion XT tris-acetate 3-8% precast polyacrylamide gel, with XT tricine running buffer (Bio-Rad Laboratories). These samples were run with laemmli buffer (Bio-Rad laboratories) with 5% beta mercaptoethanol, 10μl cell lysate, 20μl of supernatant.

2.11.2 Deglycosylation experiments

Cell lysate samples (10μl) were digested with PNGase F (New England BioLabs) according to the manufacturer’s instructions. The digested and undigested products then underwent gel electrophoresis on 3-8% Tris-acetate gels. Similarly cell lysate and supernatant samples (10μl) were digested with the restriction enzyme Endoglycosidase H (New England Biolabs) according to the manufacturer’s instructions.

2.12 Purification of ADAMTS13

ADAMTS13 expressed by stable line cells was purified using a Fast Performance Liquid Chromatography (FPLC) system (GE Healthcare) using a 5ml HisTrap HP affinity column (GE Healthcare). The column was first stripped of its Ni2+ ions using stripping solution (20mM sodium phosphate, 0.5M NaCl, 50mM EDTA, pH 7.4) followed by washing (20mM sodium phosphate, 20mM imidazole, 0.5M NaCl, pH 7.4). The column was then recharged with zinc using 100mM ZnCl solution. The column was stripped and recharged with Zn2+ as during the purification procedure in previous experiments it was found that the Ni2+ within the column displaced the ADAMTS13 Zn2+ ions.

The column was washed with binding buffer (20mM HEPES, 20mM imidazole, 500mM NaCl, pH 7.4). After this 20mM imidazole was added to the stable line supernatant to prevent non-specific binding and this was then passed through the column. Following this, elution buffer was passed through the column (20mM HEPES, 500mM imidazole, 0.5M NaCl, pH 7.4). The eluted protein was collected in

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2.5ml fractions and the fractions containing ADAMTS13 were identified by an increased absorbance of UV light.

Fractions containing the eluted protein were pooled together and the buffer exchanged (20mM HEPES, 150mM NaCl, pH 7.4) using an Ecno-Pac 10G column (Bio-Rad).

After purification the purity was confirmed using SDS electrophoresis and the quantity of ADAMTS13 purified was measured using an Imubind ADAMTS13 antigen ELISA (Sekisui Diagnostics).