This worked was supported by grants from National Health and Medical Research Council (GNT1125033 and GNT1140187) and Australian Research Council (DP170103790) to I.K.H.P.

1. Induction of Apoptosis
<ol>
	<li>Centrifuge cell sample at 300 x g for 5 min and discard supernatant to remove any pre-existing cell debris. 
	NOTE: When using adherent cells, seed cells in advance and wash with 1x phosphate-buffered solution (PBS) prior to apoptosis induction.</li>
	<li>Determine cell number and collect cells. 
	NOTE: Depending on the assay post-isolation, we recommend a starting cell number of at least 1 x 107 cells.</li>
	<li>Resuspend in complete media (respective medium containing 10% (vol/vol) fetal calf serum, 50 IU/mL penicillin, 50 &#181;g/mL streptomycin mixture) for a final concentration of 1 x 106 cells/mL.</li>
	<li>Aliquot ~2 x 106 cells per well of a 6 well plate.</li>
	<li>To induce apoptosis, remove the plate lid and irradiate cells at 150 mJ/cm2 using a UV irradiator. This should take approximately 30-60 s. 
	NOTE: Prior to irradiation, ensure that ~0.5 x 106 cells are retained for the &#39;Untreated&#39; cell control. 
	NOTE: Apoptosis can also be induced via other methods such as anti-Fas or serum starvation6.</li>
	<li>Incubate at 37&#176;C, 5% CO2 for 2-8 hours, depending on the cell line.</li>
	<li>Using a bench top light microscope, visualize cells to confirm the presence of apoptotic morphologies, such as blebbing, apoptopodia formation, and ApoBD formation. 
	NOTE: 40X magnification is sufficient</li>
	<li>Using a P1000 pipette, pipette and collect apoptotic samples.</li>
	<li>Wash the plate with 1x PBS and combine with remaining sample to ensure maximum yield.</li>
	<li>Collect ~1/10th for the &#39;Whole Apoptotic Sample&#39; (WAS) control.</li>
	<li>Collect the &#39;Untreated&#39; sample.</li>
	<li>Centrifuge both the WAS and Untreated samples at 3,000 x g for 6 min.</li>
	<li>Resuspend in 1 mL of 1x PBS and set aside on ice.</li>
	<li>For ApoBD isolation, continue to either step 2 or 3 with the remaining apoptotic sample.</li>
</ol>
2. ApoBD Isolation via FACS
<ol>
	<li>Centrifuge the entire sample at 3,000 x g for 6 min.</li>
	<li>Remove the majority of the supernatant without disrupting the pellet.</li>
	<li>Resuspend in a staining solution containing 1 mL of 1x A5 binding buffer, 75 &#181;L of A5-FITC, and 2 &#181;L of TO-PRO-3 per 1x107 cells.</li>
	<li>Incubate sample in the dark at room temperature for 10 min.</li>
	<li>Add 1-2 mL of 1x A5 binding buffer and centrifuge sample at 3,000 x g for 6 min to remove excess stain. 
	NOTE: For mixed cell populations or tissue samples, perform an antibody staining step using a combination of cell type-specific markers in 1x A5 binding buffer and incubate on ice for 20 min (or as per manufacturer&#39;s protocol) before centrifugation at 3,000 x g for 6 min.</li>
	<li>Resuspend sample pellet in 3 mL of FACS buffer (1x PBS, 1x A5 binding buffer, 10% FSC, 2 mM EDTA) per 1x107 cells.</li>
	<li>Filter through a 70 &#181;m cell strainer into a round-bottom, polypropylene (flow cytometry) tube and keep samples on ice and in the dark.</li>
	<li>Turn on the FACS machine and perform standard set up using a 100 &#181;m nozzle, perform the drop delay, and ensure a stable stream.</li>
	<li>Load the sample and set acquisition speed to ~1000 events/s.</li>
	<li>Adjust FSC, SSC, APC (TO-PRO-3) and FITC (A5) voltages and position events within the FACS plots to ensure populations can be clearly separated.</li>
	<li>Record 20,000 events.</li>
	<li>Set up a gating strategy as in part 4.</li>
	<li>In the sort layout, add the final ApoBD gate as the desired sorting population.</li>
	<li>Begin acquiring the sample and perform a test sort by collecting 5,000-10,000 ApoBDs into a new tube containing ~250 &#181;L FACS buffer.</li>
	<li>Perform a system back flush and load sorted ApoBDs.</li>
	<li>Acquire and record test-sort ApoBDs.</li>
	<li>Check that the ApoBD purity is ~99% by comparing FSC (y-axis) vs A5 (x-axis events). 
	NOTE: A5 staining may reduce slightly when re-analyzing samples due to laser bleaching.</li>
	<li>Once high purity is achieved, load original sample and continue sorting until the desired number of ApoBDs has been obtained. 
	NOTE: If necessary, dual sorting can be performed to simultaneously isolate ApoCells and ApoBDs. 
	NOTE: When sorting over a long period of time, we recommend incubating the collection tube at 4 &#176;C.</li>
	<li>Once sorting is complete, collect a small portion of post-sort ApoBDs, post-sort ApoCells, Untreated, and WAS to validate apoptosis and confirm post-sort purity. 
	NOTE: Although the test-sort and post-sort purity should not differ significantly, this is based on the stream settings and stability.</li>
</ol>
3. ApoBD Isolation via Differential Centrifugation
<ol>
	<li>Centrifuge the remaining apoptotic sample at 300 x g for 10 min.</li>
	<li>Collect the supernatant, leaving ~500 &#181;L to avoid disrupting the cell pellet, and add into a new 15 mL conical tube.</li>
	<li>Remove the remaining 500 &#181;L and resuspend the cell pellet in 2 mL of 1x PBS (this represents the &#39;ApoCell-enriched fraction&#39;.</li>
	<li>Centrifuge the collected supernatant for 20 min at 3,000 g.</li>
	<li>Check for a pellet and carefully remove the supernatant (the supernatant may contain small extracellular vesicles including microvesicles and exosomes).</li>
	<li>Resuspend the pellet in 1 mL of 1x PBS (this represents the &#39;ApoBD-enriched&#39; fraction)</li>
	<li>Collect 100 &#181;L of each Viable, WAS, ApoCell-enriched, and ApoBD-enriched samples in a new round-bottom, polystyrene (flow cytometry) tube.</li>
	<li>Add 100 &#181;L of stain containing 2x A5 binding buffer, 1:100 A5-FITC, and 1:1,000 TO-PRO-3</li>
	<li>Incubate at room temperature for 10 min in the dark.</li>
	<li>Analyze samples by flow cytometry, using the gating strategy as described above to validate the successful induction of apoptosis and purity of the ApoBD-enriched fraction.</li>
</ol>
4. Flow Cytometry Gating Strategy
<ol>
	<li>Plot TO-PRO-3 (y-axis) against FSC (x-axis) to separate necrotic cells (TO-PRO-3high) from all other non-permeabilized events (TO-PRO-3low/intermediate).</li>
	<li>Select all non-permeabilized events and plot SSC against A5. Gate two populations including population 1 (P1), SSCintermediate/high, A5low/intermediate cells and population 2 (P2), all other events.</li>
	<li>From P2, plot TO-PRO-3 against A5 and select A5intermediate/high events to exclude all cell debris.</li>
	<li>Select all A5 positive events and plot FSC against A5. Separate ApoBDs (FSClow) from ApoCells (FSCintermediate/high). 
	NOTE: When gating ApoBDs for ApoBD isolation via the FACS-based approach, we recommend a final step by selecting ApoBDs and comparing TO-PRO-3 to A5 and selecting all events. This ensures that the final sorting gate uses fluorescence rather than FSC/SSC parameters.</li>
	<li>For viable cell analysis, select P1 and perform one of two gating strategies. For general viable cell analysis, plot FSC against A5 and select all FSCintermediate/high cells, therefore removing remaining cell debris. Alternatively, for in-depth analysis, viable cells can be separated from A5- early ApoCells by plotting TO-PRO-3 against FSC. Select TO-PRO-3low, FSCintermediate/high viable cells and TO-PRO-3intermediate, FSCintermediate/high A5- early ApoCells.</li>
</ol>

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Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184, 39-51 (1995).</li><li>Jiang, L., 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=Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry.">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nature Protocols. 11, 655-663 (2016).</li><li>Berda-Haddad, Y., 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=Sterile+inflammation+of+endothelial+cell-derived+apoptotic+bodies+is+mediated+by+interleukin-1alpha.">Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 20684-20689 (2011).</li><li>Lleo, 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=Shotgun+proteomics:+identification+of+unique+protein+profiles+of+apoptotic+bodies+from+biliary+epithelial+cells.">Shotgun proteomics: identification of unique protein profiles of apoptotic bodies from biliary epithelial cells.</a> Hepatology. 60, 1314-1323 (2014).</li><li>Atkin-Smith, G. 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The authors have nothing to disclose.

Since its early description in the 1950s, the field of apoptosis has advanced markedly, becoming a prominent research area. Despite the broad interest and extensive efforts, certain aspects of apoptosis, in particular the formation of ApoBDs, have not been well studied due to the lack of appropriate methodologies. These notably include the limitation in tracking apoptosis progression and ApoBD formation simultaneously using the traditional flow cytometry A5/PI analysis and the impurities of ApoBD isolation. We have recen...

Apoptosis, a well-studied form of programmed cell death, is required to maintain physiological homeostasis and remove potentially harmful cells within the human body1. After the induction of apoptosis, apoptotic cells (ApoCells) can undergo a series of morphological changes and disassemble into small membrane-bound vesicles termed ApoBDs. Overall, this process is known as apoptotic cell disassembly and can be divided into 3 distinct steps based on morphology2,3. Step 1 (plasma membrane blebbing) is characterized by the formation of balloon-like structures on the cell surface known as blebs4,5. Step 2 (apoptotic protrusion formation) includes the formation of long membrane protrusions such as apoptopodia, beaded-apoptopodia and microtubule spikes6,7,8. Lastly, Step 3 (ApoBD formation) includes the fragmentation of the apoptotic protrusions and/or ApoCells to generate ApoBDs6,9. Previous findings have suggested a role of ApoBDs in aiding apoptotic cell clearance and mediating intercellular communication. For example, it is proposed that the fragmentation of an ApoCell into ApoBDs may generate small &#39;bite-sized&#39; pieces that could be easily removed by surrounding phagocytes2,10,11. Furthermore, ApoBDs may harbor a series of biomolecules such as DNA, RNA and proteins, which may be trafficked to surrounding cells to facilitate cell-cell communication12,13,14. To functionally investigate these processes, it is vital to confirm three key parameters including (i) validation of apoptosis induction and ApoBD formation, (ii) isolation of ApoBDs, and (iii) confirmation of ApoBD purity.
Previously, a number of methods including flow cytometry and electron microscopy have been used to study apoptosis and ApoBDs15,16,17,18. However, ApoBD detection and quantification are often difficult or overlooked. For instance, the most routinely-used flow cytometry-based apoptosis assay employs annexin V (A5, a protein that binds the externalized &#39;eat-me&#39; signal phosphatidylserine (PtdSer)) and nucleic acid stain propidium iodide (PI)19. However, by using this universal stain combination, analysis assumes that there are only three types of cell subsets (viable cells, ApoCells and necrotic cells) in the sample. Furthermore, though considered as &#34;a gold standard&#34; by many researchers for apoptosis, flow cytometry assays and subsequent data analysis often excludes ApoBDs through an initial gating step selecting FSC/SSCintermediate-high events. Therefore, we have recently developed a novel flow cytometry assay using A5 and TO-PRO-3, another nucleic stain that can be selectively taken up by caspase 3/7-activated pannexin 1 (PANX1) channels7,20. As caspase 3-induced PANX1 activation precedes PtdSer exposure at the early stage of apoptosis, TO-PRO-3 differentially stains apoptotic and necrotic cells. In addition, this approach combined with our novel gating strategy includes all acquired events during data analysis and as a result, six cell/particle subsets are identified, including: (i) viable cells (FSC/SSCintermediate/high, A5low, TO-PRO-3low), (ii) A5- early ApoCells (FSC/SSCintermediate/high, A5low, TO-PRO-3intermediate), (iii) A5+ ApoCells (FSC/SSCintermediate/high, A5high, TO-PRO-3intermediate), (iv) necrotic cells or late ApoCells (FSC/SSCintermediate/high, A5high, TO-PRO-3high), (v) ApoBDs (FSC/SSClow, A5intermediate, TO-PRO-3low/intermediate), and (vi) debris (FSC/SSClow, A5low, TO-PRO-3low)20. Our approach emphasizes the importance of analyzing all cells/particle subsets and, more importantly, the separation of ApoBDs from cells and debris20. Thus, this approach demonstrates an efficient technique to validate the induction of apoptosis and ApoBD formation simultaneously.
Traditionally, ApoBDs have been isolated through a variety of differential centrifugation approaches whereby ApoBDs can be separated from cells or other extracellular vesicles based on density. However, such centrifugation methods are often limited by low ApoBD purity, lack of a quantification step to confirm sample purity, and/or inability to separate cell type-specific ApoBDs17,21,22. Therefore, we recently developed two approaches, a FACS-based and a new differential centrifugation-based approach which can be coupled with our previously established flow cytometry method to validate the induction of apoptosis and sample purity23. ApoBD isolation via our FACS-based approach can enrich ApoBDs to up to 99% purity, and can be coupled with a variety of cell type-specific antibodies to isolate ApoBDs from mixed cell populations, tissue samples and bodily fluids23. Furthermore, our revised differential centrifugation approach demonstrates an efficient method to isolate ApoBDs to &#62;90% purity23.
In this paper, we describe in detail our experimental procedure to validate apoptosis induction, and to detect and quantify ApoBD formation. The ApoBD isolation workflows using FACS-based and differential centrifugation-based methods are also elaborated and compared. The representative data demonstrate that the described methodology provides an effective cutting-edge tool for future ApoBD studies.

<table border="0" cellpadding="0" cellspacing="0" style="width:1309px;" width="1309">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">-</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">RPMI 1640 medium</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">22400-089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Penicillin-streptomycin mixture</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FSC</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">10099-141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x PBS</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexin V FITC</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO-PRO-3 iodide</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:119px;">T3605</td>
			<td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Annexin V binding buffer</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">556454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:119px;">1001710526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Centrifuge tube (15 mL)</td>
			<td style="width:149px;">Cellstar</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Microcentrifuge tube (1.5 mL)</td>
			<td style="width:149px;">Sarstedt</td>
			<td style="width:119px;">72.690.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Tissue culture incubator (37 &#176;C, 5% CO2)</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Centrifuge</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:119px;">392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FACS Diva 6.1.1 software</td>
			<td style="width:149px;">BD Bioscience</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">FlowJo 8.8.6 software</td>
			<td style="width:149px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;UV Stratalinker 1800</td>
			<td style="width:149px;">Stratagene</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection and isolation of apoptotic bodies to high purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

Using the procedure outlined here, THP-1 monocyte apoptosis was induced and ApoBDs were detected and isolated via either a FACS-based or a differential centrifugation approach (Figure 1). Firstly, apoptosis was induced by UV irradiation and samples were collected after 2-3 h of incubation when cells exhibited apoptotic morphologies, including blebbing, apoptotic membrane protrusion formation and the generation of ApoBDs6. A TO-PRO-3 an...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.5K Views.  La Trobe University. A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity. 

Video: Detection and Isolation of Apoptotic Bodies to High Purity

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...