This research was sponsored by an intramural grant awarded for A.A. This work was supported by a CTSA grant from NCATS awarded to the University of Kansas Medical Center for Frontiers: The Heartland Institute for Clinical and Translational Research (#UL1TR606381). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or NCATS. We are thankful for The University of Kansas Medical Center ALS clinic personal for obtaining IRB approval for blood sample collection from healthy volunteer and ALS patients. The authors thank Emre Agbas for proofreading of the manuscript.

All protocols concerning the processing of human platelets follow the guidelines of both the University of Kansas Medical Center and Kansas City University of Medicine and Biosciences IRB committees.
1. Preparation of buffers and reagents
NOTE: Prepare all samples as per the manufacturer&#39;s guidelines. Wear personal protection equipment (lab coats, gloves, and goggles) during this procedure.
<ol>
	<li>Prepare citrate wash buffer by combining 0.941 g of sucrose (11 mM final concentration), 6.4 mL of 5 M NaCl (128 mM final), 5.4 mL of 0.2 M NaH2PO4 (4.3 mM final), 9.4 mL of 0.2 M Na2PHO4 (7.5 mM final), 0.352 g of sodium citrate (4.8 mM final), and 0.115 g of citric acid (2.4 mM final). Adjust the total volume to 250 mL with ddH2O. Filter through a 0.45 &#181;m filter disk and adjust the pH to 6.5. Store up to 1 year at 4 &#730;C. Bring the solution room temperature (RT) before use22.</li>
	<li>Prepare the rupture buffer by combining 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-Cl (pH 7.4) in a 100 mL final volume. Store up to 1 year at 4 &#730;C. Add 2 &#181;L of phosphatase inhibitors cocktail (1:1000 final) and 1 &#181;L of protease inhibitors cocktail (1:2000 final) into 2 mL of rupture buffer. Keep on ice until use. Discard the unused rupture buffer.</li>
</ol>
2. Platelet isolation
<ol>
	<li>Collect 8&#8211;10 mL of human blood in yellow-cap blood collection tube containing acid-citrate-dextrose (ACD) solution (75 mM trisodium citrate, 124 mM dextrose, and 38 mM citric acid, pH = 7.4; ACD:blood = 1:9). Gently mix the tube content 5x&#8211;6x inverting by hand.</li>
	<li>Centrifuge the tubes at 200 x g in a swinging bucket rotor for 20 min at RT.</li>
	<li>Collect platelet-rich plasma (PRP) (~3&#8211;4 mL) into a 15 mL conical bottom tube and leave approximately 0.5 mL of the PRP from the buffy coat (hazy-looking fraction) to avoid contamination. If any red blood cell contamination occurs, repeat this step.</li>
	<li>Centrifuge the PRP samples at 1,200 x g for 15 min at RT.</li>
	<li>Wash the platelet pellets (P1) by gentle resuspension in 1 mL of citrate wash buffer and pellet by centrifugation at 1,200 x g for 15 min at RT.</li>
	<li>Save the pure platelet pellet. Discard the supernatant.</li>
	<li>Resuspend the platelet pellets in 600 &#956;L of the rupture buffer containing inhibitor cocktails.</li>
	<li>Sonicate the platelet suspension using a sonicator. Place the sample in a mini ice bucket. Set the sonicator at setting 3 for 20 s in continuous mode. 
	NOTE: Make sure to clean the probe with 10% bleach followed by distilled water.</li>
	<li>Centrifuge the sonicated samples at 20,000 x g for 30 min at 4 &#176;C to remove membranous fractions. Aliquot supernatants in 60 &#956;L and store at -80 &#730;C. Avoid repeated thawing/freezing cycles for the platelet cytosolic fractions.</li>
</ol>
3. Preparation for CEI
NOTE: 100 &#181;L of human platelet lysate was combined from ALS patients (n = 8&#8211;10), and healthy subjects (n = 8&#8211;10) were separately pooled and used for the assay optimization.
<ol>
	<li>Fill out in-house generated templates for the CEI layout (Table 1) and sample preparation (Table 2). The sample mixture preparation table is dynamic and will automatically calculate how much volume needs to be removed from source. 
	NOTE: When required source volume entered in dynamic Table-2, 0.1 X sample buffer volume will be automatically calculated.</li>
	<li>Pre-label 25 0.2 mL PCR tubes with capillaries #1&#8211;#25 and place them in a PCR rack. Set on ice.</li>
	<li>Pre-label 0.6 mL microcentrifuge tubes: one for each primary antibody and dilution (if needed) to be used, one for the 0.1x sample buffer, one for the luminol-S/peroxide, and one for each sample to be diluted (if needed). Place them on ice in tube rack.</li>
	<li>Take out the sample buffer, wash buffer, one plate, and a cartridge provided in the CEI separation 12&#8211;230 kDa master kit separation module.</li>
	<li>From the 4 &#176;C refrigerator, take out the antibody dilution buffer, primary antibodies, secondary antibodies, luminol, hydrogen peroxide, and standard pack. Place all reagents on ice, except the standard pack, which remains at RT. 
	NOTE: The reagents from the standard packs are lyophilized and sealed with a foil cover. These should be spun down briefly using a mini centrifuge before opening to reduce product loss. To open, the reagent tubes can either be pierced by a pipette tip or pulled back from the corner.</li>
	<li>To prepare the 400 mM DTT, add 40 &#181;L of deionized water to the clear tube containing the DTT.</li>
	<li>To prepare 40 &#181;L of fluorescent 5x master mix, add 20 &#181;L of the 10x sample buffer and 20 &#181;L of the prepared 400 mM DTT solution to the pink tube provided in kit.</li>
	<li>To prepare the biotinylated ladder, add 16 &#181;L of deionized water, 2 &#181;L of 10x sample buffer, and 2 &#181;L of the prepared 400 mM DTT solution to the white tube provided in kit. Mix gently and transfer into a 0.2 mL PCR tube for denaturing.</li>
	<li>Prepare 0.1x sample buffer by adding 1.5 &#181;L of 10x sample buffer and 148.5 &#181;L of deionized water to a 0.6 mL micro-centrifuge tube. Vortex to mix and place on ice.</li>
	<li>Prepare the desired antibody dilutions. Add antibody diluent in volumes designated to each pre-labeled micro-centrifuge tube. If volumes are identical, use reverse pipetting technique23; if not, pre-rinse the pipette tip before dispensing. 
	NOTE: In this assay, a-TDP-43 pan antibody and a-p(S409/410-2) TDP-43 antibody were used. Anti-ERK antibody was used for an internal control to make sure that assay components are working.</li>
	<li>Perform the reverse pipetting for antibody dilution as described below. Alternatively, additional information can be found in the literature24. 
	NOTE: Reverse pipetting technique is preferred when dispensing small sequential volumes of solutions23. This technique offers some advantages: (i) providing a precise volume, (ii) eliminating the reagent foaming in the tip orifice, and (iii) ideal for small volume (&#60;5 &#181;L) reagents, viscous solutions, surfactant solutions, and solutions with high vapor pressure.
	<ol>
		<li>Put a proper tip in a pipette and press the plunger down to the second stop (Step-2). Immerse the pipet tip a few millimeters into the solution. Slowly release the plunger to fill up the pipet tip with the solution while the tip is still immersed in the solution. Remove the tip from the solution and gently touch against the edge of the reagent reservoir so that excess liquid remaining on the outside of the tip is removed.</li>
		<li>Dispense the solution by pressing the plunger down to first stop (Step-1). Do not dispense the remaining solution in the tip.</li>
		<li>Empty the remaining solution in the tip to the reagent reservoir by pressing the plunger to the second stop (Step-2). Release the plunger to the ready position for the next pipetting step.</li>
		<li>Add the required antibody in volumes designated to each pre-labeled microcentrifuge tube (Table 1) Do not pre-rinse the pipet tip: add it directly to the diluent and flush the tip multiple times to remove antibody. Place the tubes on ice.</li>
	</ol>
	</li>
	<li>To prepare the CEI sample-mix, perform the steps listed below for PCR tubes labeled cap#2 through cap#25: This is in the same order as it appears in Table 1.
	<ol>
		<li>Open all tubes, add 1.6 &#181;L aliquots of fluorescent 5x sample buffer to each tube using a reverse pipetting technique, then close each PCR tube upon the addition of the 5x buffer to minimize sample loss.</li>
		<li>Open all tubes, add 0.1x sample buffer in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use a reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.</li>
		<li>Open all tubes, add protein sample in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.</li>
		<li>Briefly centrifuge all PCR tubes in a benchtop centrifuge (13,000 x g for 30 s), flick/vortex PCR tubes to mix, then repeat the centrifugation.</li>
		<li>Transfer all PCR tubes into thermocycler with a heated lid. Denature samples at defined temperature and duration (i.e., 95 &#730;C for 5 min; 70 &#730;C for 10 min). 
		NOTE: The denaturation temperature and duration need to be optimized for target protein.</li>
		<li>Repeat step 3.12.4.</li>
		<li>Return all PCR tubes to tube rack and place on ice.</li>
		<li>During the denaturing step, prepare the development solution (1:1 luminol-S:peroxide solution), then add 200 &#181;L of luminol-S and 200 &#181;L of peroxide. Place on ice.</li>
		<li>To load a CEI pre-filled plate with the sample prepared above, dispense reagents and samples into the assay plate shown in the assay layout (Figure 1). Avoid introducing air bubbles. 
		NOTE: If volumes and solution are identical, use a reverse pipetting technique. If not, pre-rinse the pipet tip before dispensing and do not expel the remainder into the plate well using the second tab stop on the pipette. A 12&#8211;230 kDa separation module may contain a color-coded plate-loading guide. Place this guide under the plate while adding reagents and samples to the well, which visually helps when sample loading. The plate-loading guide can be downloaded from company website, as well.
		<ol>
			<li>In row E, add 15 &#181;L of luminol:peroxide mix to each well. 
			NOTE: Ideally, prepare this reagent just before use and add to each well. If this is not convenient, this mixture may be prepared no more than 30 min prior to plate loading.</li>
			<li>In row D, to well D1, add 10 &#181;L of streptavidin-HRP.</li>
			<li>In row D, to wells D2&#8211;D25, add 10 &#181;L of designated secondary antibody.</li>
			<li>In row B, to each well, add 10 &#181;L of antibody diluent.</li>
			<li>In row C, to well C1, add 10 &#181;L of antibody diluent.</li>
			<li>In row C, to wells C2&#8211;C25, add 10 &#181;L of designated primary antibody.</li>
			<li>In row A, to well A1, add 5 &#181;L of biotinylated ladder from PCR tube #1.</li>
			<li>In row A, to wells A2&#8211;A25, add 3 &#181;L of the sample, PCR tubes #2&#8211;#25 into corresponding wells #2&#8211;#25.</li>
			<li>Add 500 &#181;L of wash buffer to each designated wash buffer well.</li>
			<li>Centrifuge the plate for 5 min at 1,000 x g at RT.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60638/60638fig01v2.jpg" /> 
Figure 1: Assay layout. Both primary antibody and target protein sample optimization can be performed in one assay. Capillaries 2&#8211;7, 8&#8211;13, 14&#8211;19, and 20&#8211;24 represent various protein concentration and primary antibody range. Capillary 25 represents positive control. Anti-ERK antibody was used; however, any appropriate positive control can be included. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Performing the CEI on plate 1
<ol>
	<li>First, turn on the CEI (Table of Materials) analyzer, then turn on the computer. Open the software (Table of Materials)</li>
	<li>Connect the analyzer to the online system (Table of Materials). This is a necessary step to collect the run data for trouble shooting purposes and data recovery.</li>
	<li>Click on Instrument from the top-left menu, then click Connect. Select the instrument serial number which appears as a pop-up menu. Click Connect.</li>
	<li>Select the &#34;Assay&#34; tab and select New Assay or select a saved template.</li>
	<li>Input assay parameters (Table 1) or modify template currently. Save the file name and location.</li>
	<li>Make sure that the blinking blue color indicator in the analyzer remains solid blue.</li>
	<li>Touch the silver metal button on top of the orange door to open.</li>
	<li>Carefully remove the capillary cartridge from its packaging. Insert the capillary cartridge as described by manufacturer&#39;s protocol. If correctly installed, the inside light turns to &#34;blue&#34;.</li>
	<li>Remove the protective seal from the assay plate. Visually observe the pre-filled wells for air bubbles. If observed, pop them with a small pipet tip (Long-shaft P10 pipet tip works well).</li>
	<li>Load the plate holder by placing the assay plate and close the door. In computer, click on Start button.
	<ol>
		<li>Place ice tray containing temperature sensitive reagents and samples in the dark at 4 &#730;C until ready to prepare second pre-filled plate.</li>
		<li>Leave reagents/supplies out at room temperature for the second plate.</li>
	</ol>
	</li>
</ol>
5. Performing the CEI on plate 2
NOTE: This plate is set for analyzing phosphorylated TDP-43 levels.
<ol>
	<li>Remove the ice tray being stored at 4 &#730;C and place it on the bench, 1 h before the estimated completion time of the first plate. Retrieve a second plate and cartridge.</li>
	<li>Prepare any antibody dilutions needed for the second run and store them on ice. Prepare fresh 1:1 luminol-S:peroxide solution (step 3.12.8 above).</li>
	<li>Remix and briefly re-centrifuge the sample mix and reagents needed for loading the pre-filled plate. Load the second plate according to Figure 1. Load a-p(S409-410-2) TDP-43 antibody solution in row C wells.</li>
	<li>When the first run is complete, discard the first plate and cartridge. Remove the cartridge and place in a sharp&#39;s container for disposal. Keep the stickers from the plate and cartridge for reference purposes.</li>
	<li>Close the software file and reselect the same template. The software will remember the settings from the prior run. Make any changes to the annotation as needed (i.e., changing the primary antibody).</li>
	<li>Repeat steps 4.8&#8211;4.10. Put away all 12&#8211;230 kDa master kit separation module reagents and supplies</li>
	<li>Discard left over CEI sample-mix, antibody dilutions, 0.1x sample buffer and luminol-S:peroxide mixes in accordance with university regulations.</li>
</ol>
6. Data analysis
<ol>
	<li>Once the run is completed, ensure that the following quality checks are performed.
	<ol>
		<li>In software, select the Show Standards icon and Graph View tab. Check all 25 capillaries for the peak alignments to internal fluorescent marker sizes. Correct the misalignments by selecting Force Standard or right-clicking on the incorrect peak then selecting Not a Standard. Perform this check for each new capillary.</li>
		<li>Click on Samples and the Single View icon. Select capillary #1 (biotinylated ladder) in the experiment tab. Review the peak alignments to molecular weight markers. Click on the peak in Graph View and select Remove Peak, if an incorrect selection of the peak is done by the software. 
		NOTE: As an example, the 12&#8211;230 KDa biotinylated ladder will show sizing peaks at 12 kDa, 40 kDa, 66 kDa, 116 kDa, 180 kDa, and 230 kDa. The sizing of the sample peaks will be incorrect if this step is not performed and will generating inaccurate results.</li>
		<li>View the electrophoretic movie and note if any abnormal migration occurred during the run.</li>
		<li>Derive data (e.g., peaks table, including molecular weight, peak area, peak height, and signal-to-noise [S/N]) as needed for further calculations. There are graph annotation tools located at the upper-right corner of Graph window for providing more information about the graph.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis-based+immunoassays:+principles+and+quantitative+applications.">Capillary electrophoresis-based immunoassays: principles and quantitative applications.</a> Electrophoresis. 29 (16), 3279-3295 (2008).</li><li>Fourier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+automated+capillary+nano-immunoassay-Simple+Western+assay-to+quantify+total+TDP43+protein+in+human+platelet+samples.">Development of an automated capillary nano-immunoassay-Simple Western assay-to quantify total TDP43 protein in human platelet samples.</a> Analytical and Bioanalytical Chemistry. 411 (1), 267-275 (2019).</li><li>. Compatibility, S.W.S.A.B Available from: <a target="_blank" href="https://www.proteinsimple.com/technical_library.html">https://www.proteinsimple.com/technical_library.html</a> (2017)</li></ol>

The authors declare no competing financial interest except ProteinSimple, Inc. covered the publication cost of this manuscript.

The capillary electrophoretic-based immunoassay is now the method of choice for high-throughput sample analysis and drug screenings25. Small sample volumes, well-optimized assay components, user-friendly assay platform and instrumentation, reagent expenditure, and low CV percentage are primary advantages26,27. Although there are several methods for separating proteins in different assay modalities, the antibody-based CEI described here can be adapted by small laboratories that are engaged in blood-based biomarker development. The CEI assay technology used here provides reliable, reproducible, and sensitive measurements for TDP-4328 and its phosphorylated derivative5.
The CEI system also provides a multiplexing choice of analyzing TDP-43 and its phosphorylated derivatives simultaneously and providing the direct quantification of the target protein, if purified or recombinant target protein is available. Full-length recombinant TDP-43 protein is commercially available; however, recombinant phosphorylated TDP-43 derivative is not. Since phosphorylated TDP-43 is not commercially available, a predictive phosphorylation value (PPV) was implemented to assess TDP-43 profile in ALS patients. Pan TDP-43 and phosphorylated TDP-43 amounts were permanently labelled with a fluorophore; therefore, the TDP-43 profile remains the same with or without a quantitative unit (i.e., ng/mL, pg/mL, etc.). Although determining the absolute amount of TDP-43 and its phosphorylated derivatives (i.e., P [S409-410-12] TDP-43) provides a more quantitative measurement, calculating PPV eliminates the necessity of the recombinant phosphorylated TDP-43 for standardization, since it is not commercially available.
CEI provides several checkpoints in the assay platform to accurately identify the problem in the case that an assay fails. This eliminates obstacles and provides better experimental design. The assay procedure is fully automated except for filling the sample plate. This is a significant feature compared to standard western blotting analysis. This feature provides consistency from run-to-run. Although every laboratory has unique standard operating procedures, it is important to adhere to practices that minimize human error. For example, it is critical to prepare the luminol-S/peroxide mixture just before plate loading, since adding peroxide into luminol starts the enzymatic reaction and consumes the luminol substrate. Loading samples and primary/secondary antibodies into plate wells without air bubbles are also critically important steps.
Additionally, since the plate wells are small in volume and there is no space between wells, users should use caution while pipetting, which is the most important step since everything else is automated. The loading order of the samples, antibodies, and other reagents is important for consistency of the assay (Figure 1). The process of plate preparation takes about 40&#8211;45 min. Therefore, it is recommended to first load the plate with the required assay components and prepare the luminol-S/peroxide mixture just before pipetting. This way, there is a consistent sequence of reagent adding, and consistent luminescence signal strength will be attained. It is not recommended to use an expired luminol-S/peroxide reagent, as it primarily affects the strength of the peroxide. Recent progress in introducing the split-buffer system and including the chemical and detergent compatibility range has enhanced the assay quality and produced more reproducible and predictable results. Now, a new combo-analyzer from the same manufacturer possesses a feature for analyzing the samples labeled with chemiluminescence and fluorescence conjugated antibodies in the same run. This new feature eliminates the need to consecutively run two individual plates and eliminates run-to-run variation.
The assay plates should be stored in ambient temperature. If it is chosen to keep the assay plates in a 4 &#730;C refrigerator, the plates must be taken out the night before the assay and brought to ambient temperature. Incorrectly loaded sample wells need to be extensively (4&#8211;5 times) washed with buffer provided in kit before adding the correct sample. Each primary antibody and biological samples are unique; therefore, the antibody/protein optimization should be performed before analyzing the samples for target proteins in biological fluids.
Here, the primary antibody incubation time was set for 30 min by default. If the signal is weak, users should consider increasing the primary antibody incubation time until reaching the desired signal strength without fluorescence signal burnout. For human platelets, pooled samples from patients were prepared and used for an optimization assay. The sample pooling better represents the variation among target biomolecules. It is recommended to use clear supernatants rather than total lysate or total homogenate for the CEI.
The high concentration of protein in whole platelet lysate mixture may decrease the signal-to-noise ratio (Figure 2). Repeated freeze-thaw cycles of samples should be avoided, as this adversely affects primary antibody binding. The ingredients of the lysate buffer are important, as some reagents are not compatible with CEI29. It is advised to cross-check the list of compatible reagents provided on the manufacturer&#39;s website before sample preparation. This is a limitation of the system that does not tolerate high stringency conditions for sample preparation. It is recommended to optimize the assay run parameters (i.e., primary antibody dilution, protein concentration, primary antibody incubation time, etc.) using pooled samples to subsequently analyze the individual samples.

The overall goal of CEI as described here is to provide an updated stepwise protocol for analyzing target proteins in human platelets. Assignment of a blood-based signature molecule is one of the most important tasks in the field of biomarker development in human neurodegenerative diseases, such as Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Parkinson&#39;s disease (PD), inclusion body myositis (IBM), and other protein-aggregation relevant pathologic conditions. The detection of minute amounts of such signature proteins in large volumes of blood with many interfering agents is a challenge. Therefore, the specificity, sensitivity, ability to handle large number of samples, and reproducibility of the selected method are crucial.
Human platelets can serve as a milieu to identify and assign potential biomarker proteins for neurodegenerative disease. Platelets provide the opportunity to serve as a surrogate primary cell model, which reflect some features of neuronal cells1,2,3. There are certain features that make platelets one of the preferred means to analyze biomarker candidate proteins and their chemical derivatives. First, platelets can be easily acquired using a less invasive approach by collecting blood from donors (i.e., venipuncture) or in large volumes from community blood banks. Second, platelets can be easily isolated from the whole blood with minimal preparatory work in minimally equipped laboratories4,5. Third, platelets do not have nuclei; therefore, they are a good model cell to study alterations in metabolism without transcriptional regulation. Fourth, the biomolecule content of platelets is encapsulated; therefore, the platelet microenvironment protects its contents from serum-interfering substances (i.e., proteases). Fifth, platelet-enriched plasma can be stored at room temperature for 7&#8211;8 days without losing metabolic activity. Therefore, platelets provide a working model in which external factors are minimized and controlled.
Traditional immunoassay techniques such as immunoblotting (e.g., western blotting) and enzyme-linked immunosorbent assay (ELISA) are more widely used in specific protein analysis. However, these two methods have several disadvantages, including multiple assay steps, requirement of hazardous chemicals and reagents, large sample size, issues with assay reproducibility, and inter-run data variabilities. These prompted the development of a method that is simpler with fewer steps and achievable in a relatively short period. Although the classical western blot technique will remain a popular laboratory method, its multi-step procedure, supplies, toxic waste (i.e., acrylamide, methanol, etc.) and assay time are becoming less desirable when performing high-throughput quantitative protein analysis.
An automated CEI approach is gradually becoming a method of choice for laboratories that conduct high-throughput protein assays6. CEI eliminates the need for gels, gel electrophoresis apparatuses, membranes, electrophoresis and electro-transfer devices, and more physical handling involvements. If designed well, a CEI assay should be completed within approximately 3.5 h, including quantitative data analysis, publication quality electropherogram, and graphs with statistical analysis. Another superiority of the CEI system is its requirement of 10x&#8211;20x less protein concentration, making it ideal for use in human samples used in clinical trials7,8.
The most critical part of CEI is optimizing the assay conditions for each antibody purchased from different vendors, type of antibody (monoclonal vs. polyclonal), optimum protein concentrations, sample preparation, sample denaturation temperature, and electrophoresis voltage applied on the capillaries. We have developed a single-assay format optimization method for the CEI that should be implemented before any new assays, which will save time and resources. This optimization step is followed by an automated quantitative assessment of both total and phosphorylated derivative of transactivation response DNA/RNA binding protein (TARDP). Due to its size (43 kDa), the acronym TDP-43 will be used throughout this paper. Here, TDP-43 protein in human platelet lysate obtained from ALS patients are assessed to help develop predictive phosphorylation value (PPV) as a potential prognostic biomarker.
TDP-43 is a new potential disease biomarker candidate for ALS. TDP-43 is an omnipresent protein in all nucleated cells; therefore, the functions of TDP-43 during various normal cellular events and in neurodegenerative disease have been investigated9,10,11,12,13,14. Although TDP-43 is a nuclear protein15, it has the ability to shuttle in and out between the nucleus and cytoplasm due to the presence of nuclear localization and nuclear export sequences16,17,18,19. Cytoplasmic TDP-43 is involved in various cellular events, such as mRNA stability and transport, the stress response, mitochondrial function, autophagosome20. However, not much is known about the role of phosphorylated derivatives of TDP-43 other than their involvement in the pathogenesis of neurodegenerative disease21.
This protocol illustrates how to optimize the assay conditions to analyze the contents of TDP-43 and its phosphorylated derivative in platelets using the CEI approach. Since phosphorylated TDP-43 is not commercially available, it is proposed to use a predictive phosphorylation value (PPV) to assess TDP-43 profiles in ALS patients. This CEI system utilizes a small volume of sample mixture (2.5&#8211;3.0 &#181;L per capillary). Total assay volume set-up is 8.0 &#181;L per capillary based on the manufacturer&#39;s protocol; hence, researchers can utilize one sample mixture preparation for two separate runs. The manufacturer designed the assay protocol so that any pipetting errors are minimized, if not entirely eliminated. The 24 individual human platelet lysate sample mixtures are divided into half-volumes (i.e., 2.5&#8211;3.0 &#181;L per sample) and consecutively analyzed those by two different antibodies within ~7 h. The CEI system described here provides a desirable high-throughput assay modality. Users need to test antibodies from different vendors and sample preparation modalities for the target protein before performing large-scale screening.

<table><tbody><tr><td>12-230 kDa Separation kit</td><td>ProteinSimple</td><td>SM-W004</td><td>Contains pre-filled assay plate and 25-channel capillary cartridge</td></tr><tr><td>3000G Thermocycler</td><td>Techne</td><td>FTC3G/02</td><td>We used this thermocylcer for heating the sample mix</td></tr><tr><td>Anti-Mouse detection kit</td><td>ProteinSimple</td><td>042-205</td><td>Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker</td></tr><tr><td>Anti-P(S409-410) TDP-43 antibody</td><td>ProteinTech</td><td>22309-1-AP</td><td>Primary antibody that recognizes phosphorylated TDP-43</td></tr><tr><td>Anti-P(S409-412) TDP-43 antibody</td><td>CosmoBio-USA</td><td>TIP-PTD-P02</td><td>Primary antibody that recognizes phosphorylated TDP-43</td></tr><tr><td>Anti-Rabbit detection kit</td><td>ProteinSimple</td><td>DM-001</td><td>Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker</td></tr><tr><td>Anti-TDP-43 (pan) antibody</td><td>ProteinTech</td><td>10782-2-AP</td><td>Primary antibody that recognizes whole TDP-43 protein</td></tr><tr><td>Compass for SimpleWestern (SW)</td><td>ProteinSimple</td><td>Ver.4.0.0.</td><td>Compass for SW is the control and data analysis application for SimpleWestern instruments</td></tr><tr><td>Sonic Dismembrator; Model100</td><td>Fisher Scientific</td><td></td><td>Sonicator. Used to rupture the cell membrane. This model is discontinued (Model XL2000-350)</td></tr><tr><td>Table top centrifuge</td><td>Eppendorf</td><td>22625004</td><td>Model# 5810 with swinging plate bucket</td></tr><tr><td>&lt;em&gt;Wes&lt;/em&gt; analyzer</td><td>ProteinSimple</td><td>55892-WS-2203</td><td>Performs the capillary gel electrophoresis</td></tr></tbody></table>

use of capillary electrophoresis immunoassay to search for potential biomarkers of amyotrophic lateral sclerosis in human platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Optimization of platelet cytosolic protein concentration and primary antibody titration 
It is important to establish a linear dynamic range of platelet cytosolic proteins in the assay, since changes in signal are directly proportional to changes in protein in the platelet cytosol. Use of whole platelet lysate mixture in the assay may reduce the signal intensity of the target proteins (TDP-43 and P(S409-412) TDP-43) and contribute to a high background signal. Therefore, in this assay, the clear supernatant was used (cytosolic fraction) after rupturing platelets (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60638/60638fig02v2.jpg" /> 
Figure 2: Signal clarity depends on the sample quality. (A) Whole platelet lysate homogenate interferes with anti-TDP-43 antibody binding; therefore, a noisy electropherogram was observed. (B) Platelet cytosolic fraction was obtained from subjecting the whole lysate to centrifugation (16,000 x g for 30 min). Most of the membranous proteins were removed; hence, anti-TDP-43 antibody binding to TDP-43 protein was enhanced (blue line trace). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A linear dynamic range for platelet cytosol protein concentration was established at 0.2&#8211;0.8 mg/mL. An assay template was adopted so that both protein concentration and primary antibody titration were able to be performed in one assay (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60638/60638fig03v2.jpg" /> 
Figure 3: Linear dynamic range for platelet cytosol protein concentration. (A) Both protein concentrations and antibody titrations were optimized in one plate during the same run. (B) The linear working range (0.2&#8211;0.8 mg/mL) for protein was established. A 0.5 mg/mL protein load was labeled by a-ERK antibody as the positive control (capillary 25). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It should be noted that glycerol content in the sample preparation tube should be less than 20% (final), otherwise the high glycerol concentration will adversely affect the primary antibody binding.
Determining optimum exposure time 
In the older version of the software, the optimum exposure time had to be determined by plotting peak area against protein concentration (mg/mL). The new version provides a new tool named the high dynamic range (HDR) detection profile (Figure 4). Using the image panel provided the option to view all exposure times (i.e., 5 s, 15 s, 30 s, 60 s, 120 s, 240 s, 480 s) together. Computer software analyzed all exposure times and automatically identified the best exposure time (HDR). HDR detection profile delivered a significantly wider dynamic range due to the greater sensitivity of CEI, which means better detection and quantitation over a larger sample concentration range. However, users still have the option to choose any exposure time that satisfies the experimental goal. Using this feature, optimum exposure time was found for TDP-43 protein. The peak represents the optimum exposure time (Figure 4A). A single exposure time (4 s) was defined for this antibody after reviewing all nine exposure times ranging between 1&#8211;512 s (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60638/60638fig04v2.jpg" /> 
Figure 4: High dynamic range (HDR) detection profile for a target protein. (A) TDP-43 protein peak represents the optimum exposure time for the target protein. a-TDP-43 Ab titration was 1:300, and platelet cytosol protein concentration was 0.5 mg/mL. The software-defined blue line indicates the optimum exposure time. (B) This figure represents a user-defined single exposure time (4 s) after reviewing all nine exposure times ranging between 1&#8211;512 s. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
TDP-43 levels in human platelet cytosol of ALS patients 
A blood base biomarker development was performed. Using optimized assay conditions, platelet lysate cytosolic fractions obtained from ALS patients were analyzed using two sets of antibodies (i.e., anti-TDP-43 [Pan] antibody, an antibody that recognizes phosphorylated derivatives of TDP-43 protein; here, a-P [S409/410-2] TDP-43 was used). In this demonstration, disease-specific TDP-43 and its phosphorylated derivative are presented (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60638/60638fig5a.jpg" /> 
Figure 5: A representation of predictive phosphorylation value (PPV) of TDP-43. Absolute amount of phosphorylated TDP-43 and pan TDP-43 alone did not show much difference between the ALS and control groups. However, PPV indicated a slight increase in the ALS cohort, although there was no statistical difference between the two groups due to insufficient numbers of subjects (ALS = 25, control = 27). A low Cohen&#39;s d value between the means of ALS and control group showed a low effect size between the two groups due to small sample size (control = 25, ALS = 27). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Total TDP-43 was quantified using the calibration curve (Figure 6)
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60638/60638fig06v2.jpg" /> 
Figure 6: Standard calibration curve. Commercially purchased recombinant TDP-43 protein concentrations were used to construct a standard curve. Each data represents the average of triplicates. The protein band intensities were concentration dependent (Inset). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The quantification of phosphorylated TDP-43 protein was not possible due to commercially unavailability of this protein. Instead, a predicted phosphorylation value (PPV) was established that defines the percent of the phosphorylated species of TDP-43. PPV was determined from two sequential CEI assays for the same sample using the following equation.
<img alt="Equation 1" src="/files/ftp_upload/60638/60638equ01B.jpg" />
Intra- and inter-run assay variability were tested in pooled human ALS platelet cytosolic fractions (Figure 7)
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60638/60638fig07v2.jpg" /> 
Figure 7: Assay variations. (A) Two capillaries loaded with the same sample were analyzed by CEI, and intra-run assay variation was calculated as CV% = 14.9. (B) The same sample was analyzed on three different assay days and in three different assay runs. Inter-run assay variation was calculated as CV% = 18.7. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig7alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although intra-run (capillary-to-capillary variation) coefficient variation values fell in the acceptable range (CV% = 14.9), the inter-run assay value was relatively high (CV% = 18.7). It is interpreted that this variation may be due to using capillary cartridges and sample plates from different lots. It is recommended that reproducibility studies should be performed in CEI components that have the same lot number.
Table 1: CEI assay plate loading template. <a href="https://cloudflare.jove.com/files/ftp_upload/60638/Table_1_Wes_Assay_Layout_single_sheet.xlsx" target="_blank">Please click here to download this table.</a>
Table 2: Interactive sample mixture preparation template. After entering the stock protein concentration from unknown samples, interactive cells will automatically calculate how much volume needs to be used for preparing the sample mix. <a href="https://cloudflare.jove.com/files/ftp_upload/60638/Table_2_Wes_Assay_Sample_mix_preparation_template_UD.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets at JoVE.com

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

use-capillary-electrophoresis-immunoassay-to-search-for-potential

Research

JoVE Journal

Neuroscience

7.2K Views.  Kansas City University of Medicine and Biosciences. Dit protocol kan worden gebruikt om zowel eiwit- als primaire concentraties van antilichamen in één testplaat te optimaliseren en om twee tests uit te voeren met behulp van één monsterpreparaat. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt 2 1/2 dagen lab werk inclusief data-analyse, worden voltooid in 3 1/2 uur. Deze techniek biedt een hoge doorvoertest voor het ontwikkelen van een bloedgebaseerde biomarker voor ALS en is een ideale methode voor het uitvoeren va...

Video: Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Environment

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Bioengineering

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Medicine

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Dynamic Multiparameter Platelet Function Assessment Using a Capacitive Biosensor

Platelets play a fundamental role in blood clotting through a series of regulated responses, including adhesion, spreading, granular secretion, aggregation, and cytoskeletal contraction. However, current assays are limited to partial analysis of platelet function under non-physiological conditions. Thus, an improved assay that reflects the dynamic and multifaceted nature of platelet function in physiological settings is necessary. In this context, a novel approach is introduced to measure several key parameters related to platelet function in a more physiologically relevant ex vivo semi-rigid microenvironment compared to traditional assays. This method utilizes an advanced electrical biosensor, the membrane capacitance sensor (MCS), which provides unique insights into the clotting process through three distinct readouts. These readouts are highly sensitive to variations in platelet count, stimulation intensity, and specific activation pathways. As a purely electrical sensing platform, the MCS demonstrates significant potential as a diagnostic tool for detecting primary hemostatic function disorders, evaluating the efficacy of therapeutic treatments, and advancing the broader understanding of the roles of platelets in hemostasis and thrombosis.

A protocol for a novel dynamic multiparameter platelet functional assay using a capacitive biosensor is presented here. This approach, designed within a semi-rigid microenvironment to enhance physiological relevance, provides three output parameters sensitive to platelet count, stimulation strengths, and activation pathways.

Platelets play a fundamental role in blood clotting through a series of regulated responses, including adhesion, spreading, granular secretion, ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Biology

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Imaging Single Platelet Migration Using a Three-Layer Coating System

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to prevent bleeding after injury, but they also fight infections and maintain vascular integrity during inflammatory diseases. While hemostatic plugs require the collective activation and aggregation of platelets, their role in protecting inflamed blood vessels is performed at the single-cell level. In this context, recent data have shown that platelets can migrate autonomously, a process dependent on the mechanosensing of their adhesive environment. Here, a detailed protocol for imaging single platelet migration is presented, utilizing a three-layer coating system consisting of a poly-L-lysine graft poly(ethylene glycol) (PLL-PEG)-biotin backbone (1), a fluorescent avidin linker (2), and biotin-cyclic Arg-Gly-Asp (cRGD) (3) as the platelet integrin-binding motif. This reductionist approach allows precise control of substrate adhesion properties and serves as a simple, standardized in vitro assay to study the mechanisms underlying platelet migration. The results indicate that migrating platelets binding to cRGD exert forces capable of disrupting the avidin-biotin bond. Furthermore, the density of biotin-cRGD significantly influences both platelet spreading and migration.

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin bond.

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to ...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

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