The authors acknowledge Meike Schaffrinski for excellent technical assistance. We would like to thank the following people for their help in the collection of peripheral blood samples (all from University Medical Center G&#246;ttingen): Henrietta Vida (Dept. of Transfusion Medicine), Kia Homayounfar, Lena-Christin Conradi (Dept. of General, Visceral and Pediatric Surgery), Leila Siam, Bawarjan Schatlo (Dept. of Neurosurgery), Hendrik A. Wolff, Martin Canis (Dept. of Otorhinolaryngology, Head and Neck Surgery) as well as all employees of the interdisciplinary short-term oncology. We acknowledge Dirk Wenzel (Max Planck Institute for Biophysical Chemistry, G&#246;ttingen) for his help with the electron microscopy of MVs. 
The study was funded by the German Ministry of Education and Research (BMBF) project MetastaSys (grant no. 0316173) as well as the German Cancer Aid (grant no. 109615).

All experiments including human subjects have been approved by the local ethics committee (approval no. 3/2/14). For the choice of patients it should be noted that several factors such as age, sex, current therapy regimens and many more may influence MV composition in blood and therefore should be taken into consideration prior to sample collection 22,23.
1. Preparation of Plasma Samples
<ol>
	<li>Draw 1 - 2 tubes of blood per donor through a 21-gauge butterfly needle into a vacutainer blood collection tube containing EDTA (1.6 mg/mL blood). Make sure to invert the tube(s) several times to guarantee efficient blood anticoagulation. 
	NOTE: The recommended volume of blood for subsequent flow cytometry and Western Blot analysis is 5 - 15 mL. In order to prevent MV degradation and loss, blood samples should be handled &#60; 30 min after blood withdrawal.</li>
	<li>Prepare plasma samples by centrifuging the samples for 15 min at 1,200 x g at room temperature (RT).</li>
	<li>Apply a valve filter in order to help the separation of plasma (= upper layer) from remaining blood cells (= lower layer).</li>
	<li>Transfer the plasma into a 15 mL tube.</li>
	<li>Centrifuge for 15 min at 1,500 x g, RT to pellet larger cell debris and remove remaining platelets.</li>
	<li>Transfer the supernatant into a 15 mL tube and directly proceed with MV isolation or store samples for up to 6 months at -20 &#176;C. 
	NOTE: The presented protocol can also be used to isolate MVs (and Exos) from cell culture supernatants. In order to do so, cultivate cells at 60 - 80% confluency for 24 - 48 h in culture medium supplemented with vesicle-depleted FCS, and then collect the supernatant. Centrifuge for 5 min at 750 x g, 4 &#176;C to deplete residual floating cells, fill the supernatant into a new 15 mL tube and centrifuge again for 5 min at 1,500 x g, 4 &#176;C to pellet larger cell debris. This supernatant can then be used for the isolation of MVs as described in Step 2.1 - 2.16.</li>
</ol>
2. Isolation of MVs
<ol>
	<li>Transfer the plasma sample in a suitable centrifugation tube. If necessary, fill up tube with PBS in order to dilute the sample and prevent collapse of thin-walled tubes during the centrifugation procedure.</li>
	<li>Centrifuge for 35 min at 14,000 x g, 4 &#176;C.</li>
	<li>Decant supernatant, keep tubes turned upside-down and put on a paper towel. Wait 3 - 5 min until all remaining supernatant has been soaked into the towel and thereby removed from the sample.</li>
	<li>Resuspend the MV pellet in 1,000 &#181;L PBS, transfer to a 1.5 mL tube and centrifuge for 35 min at 14,000 x g, 4 &#176;C in a tabletop centrifuge.</li>
	<li>Aspirate supernatant.</li>
	<li>Resuspend the MV pellet in 50 - 500 &#181;L PBS, depending on the size of the pellet. Alternatively, lyse MVs directly, e.g., in RIPA buffer (150 mM NaCl/ 0.1% SDS/ 0.5% Na-deoxycholate/ 1% Triton X-100/ 50 mM Tris, pH 7.2) for subsequent Western Blot analysis. Store MVs at -20 &#176;C. They will remain stable for several months, but avoid repeated freeze-thaw-cycles.</li>
	<li>Optional: Determine the MV protein concentration with a protein assay (e.g., Bradford or Lowry method) in order to assess MV yields or dose MVs for subsequent experiments.</li>
	<li>If additional Exos are to be isolated from the plasma samples, decant the supernatant from step 2.3 into an ultracentrifugation tube and centrifuge for 2 h at 110,000 x g, 4 &#176;C. Decant the supernatant as described in step 2.3, resuspend Exo pellet in 1,000 &#181;L PBS and transfer into small (1.5 mL) ultracentrifugation tubes.
	<ol>
		<li>Ultracentrifuge for 2 h at 110,000 x g, 4 &#176;C, aspirate supernatant and resuspend Exo pellet in 50 - 75 &#181;L PBS or RIPA buffer.</li>
	</ol>
	</li>
</ol>
3. Characterization of MVs by Flow Cytometry
<ol>
	<li>Transfer 15 &#181;L PBS+1% vesicle-depleted fetal calf serum (FCS) in a flow cytometry tube. 
	NOTE: Vesicle-depleted FCS is prepared by centrifuging heat-inactivated (30 min at 56 &#176;C) FCS for 18 h at 110,000 x g and filtrating the supernatant through a 0.2 &#181;m filter as described previously 24.</li>
	<li>Add 5 &#181;g (in case of low yields 3 &#181;g are also applicable) of MVs in PBS.</li>
	<li>Incubate samples for 30 min at RT in order to block unspecific binding sites at the MV surface and thereby reduce background staining.</li>
	<li>Add a fluorescently labeled antibody against the protein-of-interest. Titrate the amount of antibody used for the staining prior to use in order to determine the optimal concentration and guarantee a low signal-to-noise ration. Make sure to also include one tube with unstained MVs as negative control and one tube of MVs stained with the matching isotype control antibody at the same concentration (e.g., if 1 &#181;g of antibody is used, also use 1 &#181;g of the isotype control antibody) to quantify background staining. 
	NOTE: It is also possible to perform multicolor flow cytometry by adding multiple antibodies coupled to different fluorochromes.</li>
	<li>Incubate for 20 min at RT in the dark.</li>
	<li>Add 250 &#181;L PBS and proceed with measurement of the sample using a flow cytometer.
	<ol>
		<li>In case that samples cannot be measured immediately, add 150 &#181;L PBS and 50 &#181;L 4% paraformaldehyde (PFA) to fix samples and store at 4 &#176;C. CAUTION: PFA is toxic. Use gloves and suitable personal protective equipment.</li>
	</ol>
	</li>
	<li>Reduce the threshold of the flow cytometer to the lowest value possible and search for the MV population using a forward scatter (FSC) versus side scatter (SSC) plot in logarithmic scale. Gate on the MV population and evaluate the fluorescent signal in a corresponding histogram.</li>
</ol>
4. Characterization of MVs by Western Blotting
<ol>
	<li>Resuspend the MV pellet directly in a suitable lysis buffer (e.g., RIPA buffer).
	<ol>
		<li>In case the MV pellet has already been resuspended in PBS, dilute it at least 1:1 in a suitable lysis buffer (e.g., RIPA buffer).</li>
	</ol>
	</li>
	<li>Determine the protein concentration of the MV sample, e.g., by Lowry assay.</li>
	<li>Prepare 10 - 20 &#181;g of MVs in 22.5 &#181;L RIPA buffer. Then add 7.5 &#181;L 4x Laemmli loading buffer and heat for 5 min at 95 &#176;C.</li>
	<li>Load samples on a polyacrylamide gel and perform electrophoresis and immunoblotting according to standard protocols.</li>
	<li>After transferring the proteins onto the membrane, perform a Ponceau staining as a loading control according to standard protocols.</li>
	<li>Destain membrane in TBST for 5 min at RT.</li>
	<li>Block membrane for 30 min up to 1 h at RT in 5% BSA in TBST.</li>
	<li>Incubate membrane with the primary antibody at 4 &#176;C overnight or for 2 h at RT.</li>
	<li>Wash the membrane with TBST 3 x 5 min.</li>
	<li>Incubate membrane with the HRP-coupled secondary antibody at a dilution of 1:10,000 in 5% BSA. Note: In case of high background signals, use milk powder instead of BSA.</li>
	<li>Wash the membrane with TBST 3x 5 min.</li>
	<li>Develop membrane with an ECL detection reagent and detect signals on chemiluminescence films or a chemiluminescence imaging system. 
	NOTE: In order to discriminate MVs from Exos, proteins like Tubulin, actinin-4 or mitofilin can be used which should mainly be present on MVs 16,25. Pay attention that most tetraspanin antibodies (e.g., CD9, CD81), used as markers for Exos, do not work under reducing conditions and should therefore be prepared in non-reducing loading buffer followed by heating for 10 min at 70 &#176;C.</li>
</ol>

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The authors have nothing to disclose.

Recent studies on EVs in blood have demonstrated that EV composition and counts change during the cause of several diseases. Therefore, the analysis and further characterization of these EVs are of high interest to further assess their potential use as disease biomarkers for diagnosis and prognosis or to evaluate therapy responses. The protocol we present here allows the isolation of vesicles with a diameter of up to 600 nm which do not contain any cell organelles. These observations are in line with the current definition of MVs and exclude the presence of apoptotic bodies 2. Using Western Blotting, we were able to demonstrate that the isolated MVs show a high expression of Tubulin, while the tetraspanins CD9 and CD81 that are often used as Exo markers were only slightly expressed. This confirms that MVs differ from Exos and fits to recent in-depth characterization and comparison of both EV populations by proteomics 25.
During acquisition of blood samples, it is critical to keep the time between venipuncture and plasma preparation as short as possible in order to prevent MV degradation. Moreover, prolonged storage of blood samples could lead to the activation of blood cells causing enhanced MV shedding and ultimately apoptosis which results in the release of apoptotic bodies. Another important consideration for MV isolation is to prevent contaminations of MV preparations with plasma proteins or smaller Exos. Therefore, it is critical to remove as much of the supernatant as possible after spinning down the MVs at 14,000 x g. Since the pellet is normally visible and tightly attached to the wall of the tube, the supernatant can be easily removed with a pipette tip. In contrast to Exos which tend to form aggregates during preparation by high speed ultracentrifugation and are often hard to resuspend, this problem does not occur with MVs.
Our study shows that it is possible to characterize MV subpopulations present in blood by flow cytometry. Although the detection limit of most flow cytometers is around 200 - 300 nm, MVs were reproducibly measured in donor as well as cell culture samples with the same parameters of analysis and gates that clearly allowed their distinction from background signals. It is important to verify prior to the measurements that the PBS used for the analyses does not contain any contaminating particles that could cause a high background during flow cytometry (Figure 3). Although some smaller MVs might not be captured in a flow cytometric approach, we detected MVs from all major blood cell populations (e.g., platelets, red blood cells, leukocytes, endothelial cells). In our analyses we used standard markers for the different blood cells that have been previously found on MVs 26-29. It should be noted that in order to obtain the best possible results by flow cytometry, the amount and concentration of all antibodies should be titrated on a MV sample expressing the antigen of interest. If specific MV subpopulations in blood shall be identified with higher specificity, it is possible to perform double staining against two different antigens present on the respective MVs and only consider all double positive MVs for subsequent analyses 23. Currently, there are efforts to define a standard range of MV subpopulations in blood of healthy individuals 23,30. These studies have already shown that platelet-derived MVs constitute the largest population of MVs in blood which is in correspondence with our observations.
One advantage of flow cytometry to characterize MV samples is that this method is already well established for diagnostic purposes in most clinical centers which would allow the possible use of MVs as biomarkers in everyday clinical diagnostics. Previous studies on EVs in blood which have mostly focused on smaller Exos, rely on either specific sorting procedures to selectively analyze the desired Exo target population 31,32 or require a time-consuming (2 days) isolation process with coupling of Exos to Latex beads prior to analysis 18. Our own unpublished observations suggest that the flow cytometric analysis of MVs from whole blood preparations even allows the detection of MVs such as tumor-derived MVs without any such selection processes.
Taken together, the protocol presented here allows the fast isolation of MVs from peripheral blood samples with standard lab equipment and their subsequent characterization using flow cytometry and Western Blotting. The whole process can be performed in around 2 h which will facilitate future studies on MV profiles in patients&#39; blood that are required to assess the potential of MVs as disease biomarkers.

In the last years many in vitro studies have demonstrated that extracellular vesicles (EVs) play an important role in intercellular communication. Living cells constantly shed vesicles which differ in size, content and biogenesis. The best studied EVs are exosomes which originate from the endosomal system where they are stored as intraluminal vesicles in multivesicular bodies. Once the latter fuse with the plasma membrane, the contained vesicles are released as exosomes (Exos, diameter 30 - 100 nm1). A second population of EV which has gained increasing attention in the last years are large microvesicles (MVs, diameter 100 - 1,000 nm) which bud off directly from the plasma membrane 2.
Both types of vesicles are surrounded by a lipid bilayer and contain nucleic acids, e.g., DNA, mRNA or miRNA 3-5, and a plethora of proteins which they can transfer to neighboring cells . While in general the protein composition of the vesicles reflects the state of the cell of origin, some proteins seem to be selectively targeted and enriched on EVs 1. A major research interest is to characterize EVs from abnormal and diseased cells in order to define specific EV signatures which might allow the use of EVs as novel biomarkers. Especially in cancer, in which often the tumor itself is not easily accessible, liquid biopsies targeting tumor-specific EVs in blood might allow monitoring of therapy responses or help characterizing the primary tumor without the need for invasive procedures 6.
Indeed, EVs have already been successfully isolated from various body fluids including urine 7, CSF 8, breast milk 9 or blood 10. Several studies identified changes in EV counts and composition in different human diseases. For example, in sepsis patients the number of pro-coagulant MVs is significantly increased compared to healthy individuals 11. Also in patients with severe cerebral malaria an increase in total MVs in blood can be observed and counts of platelet-derived MVs correlate with coma depth and thrombocytopenia 12. Other studies report elevated numbers of endothelium-derived vesicles in patients with systemic lupus erythematosus or heart failure and in the case of the latter, this correlates with a higher probability of cardiovascular events 13,14.
Especially in cancer, EVs in blood are currently discussed as novel biomarkers with diagnostic and prognostic value. Levels of MVs expressing tumor-associated proteins such as MUC1, EGFR or FAK seem to be elevated in the blood of breast cancer patients 15,16. Also for Exos, recent studies have shown that blood-derived Exos carrying tumor-specific antigens such as Glypican-1 for pancreatic cancer or Del-1 for breast cancer allow early disease detection with high specificity and sensitivity 17,18. Additionally, serum-derived tumor Exos may contain DNA which can be used for detection of mutations such as KRAS and p53 which suggests their use for therapy prediction 19. Recent advances have shown that analysis of Exos in blood of glioblastoma patients using a specific microfluidic chip allows monitoring of therapy 20. Taken together, these findings imply that analysis of disease-specific subpopulations of vesicles gives valuable information about diagnosis, prognosis as well as therapeutic options and success.
However, isolation and analysis of Exos from blood is time-consuming, requires special lab equipment and therefore is not yet suited for routine clinical diagnostics. In contrast, MVs can be isolated much faster and, due to their larger size, can be easily analyzed by flow cytometry without the need to couple them to latex beads as it is necessary for Exos 18,21. Thus, here we present a protocol that can be used for the standardized isolation of MVs from blood samples and the subsequent characterization of MV subpopulations by flow cytometry. This protocol will allow the further study and in depth characterization of MV profiles in large patient groups which will be required in order to use MVs for everyday clinical diagnostics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>butterfly needle (21 gauge)</td>
			<td>Hospira Deutschland GmbH</td>
			<td>490P29201</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roth</td>
			<td>8076.3</td>
			<td>For 10x TBS weigh 24.2 g Tris base and 80 g NaCl, ad 1 L H2O and adjust pH to 7.6</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>For 1x TBS-T mix 100 mL 10x TBS with 900 mL H2O and add 1 mL Tween-20</td>
		</tr>
		<tr>
			<td>CD235a-PE</td>
			<td>Beckman Coulter</td>
			<td>A07792</td>
			<td>use 5&#181;l for staining</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>Beckman Coulter</td>
			<td>7782</td>
			<td>use 5&#181;l for staining</td>
		</tr>
		<tr>
			<td>CD62E-PE</td>
			<td>Biolegend</td>
			<td>336008</td>
			<td>use 0.8&#181;g for staining</td>
		</tr>
		<tr>
			<td>CD62P-PE</td>
			<td>Biolegend</td>
			<td>304905</td>
			<td>use 0.1&#181;g for staining</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>Biolegend</td>
			<td>349501</td>
			<td>1:2000 in 5%BSA in TBS-T; use non-reducing conditions for Western Blotting</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>Immunotools</td>
			<td>21270091</td>
			<td>1:1000 in 5%BSA in TBS-T; use non-reducing conditions for Western Blotting</td>
		</tr>
		<tr>
			<td>Chemiluminescence imager ImageQuant LAS-4000</td>
			<td>Fujitsu Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DC protein assay kit II</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL detection reagent</td>
			<td>GE Healthcare</td>
			<td>RPN2232</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA vacutainers for blood collection</td>
			<td>Sarstedt</td>
			<td>01.1605.001</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCanto II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fetal calf serum (FCS)</td>
			<td>Invitrogen</td>
			<td>10091148</td>
			<td>heat-inactivated (30 min, 56&#176;C)</td>
		</tr>
		<tr>
			<td>filter 0.22&#181;m</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-coupled anti-mouse secondary antibody</td>
			<td>santa cruz</td>
			<td>sc-2005</td>
			<td>use 1:10,000 in 5% milk powder in TBS-T</td>
		</tr>
		<tr>
			<td>HRP-coupled anti-rabbit secondary antibody</td>
			<td>santa cruz</td>
			<td>sc-2004</td>
			<td>use 1:10,000 in 5% milk powder in TBS-T</td>
		</tr>
		<tr>
			<td>4x Laemmli loading buffer, Roti-Load 1</td>
			<td>Roth</td>
			<td>K929.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microfuge SIGMA 1-15K</td>
			<td>Sigma Laborzentrifugen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>milk powder (Blotting-Grade Blocker, nonfat dry milk)</td>
			<td>BioRad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>multifuge 3 L-R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight LM10</td>
			<td>NanoSight Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, w/o Ca and Mg</td>
			<td>Pan biotech</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>perfusor syringe 50 mL</td>
			<td>Braun</td>
			<td>8728844F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau-S staining solution</td>
			<td>PanReac AppliChem</td>
			<td>A2935,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor Sw32.1 Ti for ultracentrifugation (6x 17 mL)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rotor TLA-120.2 for ultracentrifugation (10x 1.5 mL)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for flow cytometry (5 mL, round-bottom)</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for ultracentrifugation (15 mL)</td>
			<td>Beckman Coulter</td>
			<td>344061</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for ultracentrifugation (11* 34 mm)</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin antibody</td>
			<td>Millipore</td>
			<td>05-829</td>
			<td>1:5000 in 5%BSA in TBST</td>
		</tr>
		<tr>
			<td>ultracentrifuge Optima L-80 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge TL-100</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>valve filter Seraplas V15</td>
			<td>Sarstedt</td>
			<td>53,428</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of microvesicles from peripheral blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

In order to quantify the yield of MVs that can be isolated following the described protocol, we calculated the amount of MVs isolated from blood samples of 10 donors. The MV yield, which was assessed in a Lowry protein assay, ranged from 10 up to 30 &#181;g with a mean of 19.2 &#181;g MVs per mL blood (Table 1). The particle concentration determined by nanoparticle tracking analysis (NTA) ranged from 1.66 x 109 to 2.36 x 1010 with a mean of 5.9 x 109 particles per mL plasma sample (Table 2). Further characterization of the MVs by transmission electron microscopy revealed a population of vesicles with a diameter &#62; 100 nm that were surrounded by a lipid bilayer and did not contain any cell organelles (Figure 1A). NTA confirmed that the size of the isolated MVs ranged from 100 up to 600 nm (Figure 1B) and the mean MV size was 201 nm (Figure 1C). Staining for typical MV and Exo markers by Western Blotting demonstrated that the isolated MVs were positive for Tubulin and only showed a slight expression of CD9 and CD81, while Exos were negative for Tubulin and enriched in CD9 and CD81 (Figure 2).
Analysis of the isolated MVs by flow cytometry (Figure 3A) revealed a defined vesicle population that could be gated using the same parameters normally used for MVs isolated from cell culture supernatants and that was clearly different from the background signal obtained by the measurement of PBS + 1% vesicles-depleted FCS without addition of MVs (Figure 3B). In order to analyze the different MV populations present in blood, MVs were stained with established markers for the different blood cell populations, e.g., CD62P for platelet-derived MVs, CD45 for leukocyte-derived MVs, CD235a for red blood cell-derived MVs and CD62E for endothelial cell-derived MVs (Figure 4). This characterization showed that the percentage of MV subpopulations differed among the investigated donor blood samples, while the majority of MVs seemed to be shed by platelets in all samples.
<img alt="Figure 1" src="/files/ftp_upload/55057/55057fig1.jpg" /> 
Figure 1: Size Distribution of MVs Isolated from Peripheral Blood. A, Isolated MVs were visualized by transmission electron microscopy. B, Representative nanoparticle tracking analysis (NTA) of MVs illustrating the size distribution of the vesicles. C, The mean MV size from 10 independent preparations was measured by NTA (mean). <a href="http://cloudflare.jove.com/files/ftp_upload/55057/55057fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55057/55057fig2.jpg" /> 
Figure 2: Characterization of Isolated MVs by Western Blotting. Differential protein expression on the isolated MVs and Exos from two donors was visualized by Western Blotting. <a href="http://cloudflare.jove.com/files/ftp_upload/55057/55057fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55057/55057fig3.jpg" /> 
Figure 3: Analysis of MVs by Flow Cytometry. A, MVs are first visualized on forward (FSC) versus sidescatter (SSC) plots to gate on the respective MV population. Subsequently, these MVs are characterized for the antigen of interest by using fluorescently labeled antibodies directed against the antigen. B, Typical FSC versus SSC plots for MVs isolated from the plasma of two donors. As comparison, a typical plot for tumor cell-derived MVs from A549 lung cancer cells isolated from cell culture supernatant as well as a negative control using only PBS + 1% vesicle-depleted FCS without MVs are shown on the right. <a href="http://cloudflare.jove.com/files/ftp_upload/55057/55057fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55057/55057fig4.jpg" /> 
Figure 4: Characterization of Isolated MVs by Flow Cytometry. MVs from three donors were characterized for the expression of established blood cell markers (red) by flow cytometry. The respective isotype controls are shown in grey. <a href="http://cloudflare.jove.com/files/ftp_upload/55057/55057fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>MVs/mL blood [&#181;g]</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>29.4</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>10.3</td>
		</tr>
		<tr>
			<td>#3</td>
			<td>15.2</td>
		</tr>
		<tr>
			<td>#4</td>
			<td>31.1</td>
		</tr>
		<tr>
			<td>#5</td>
			<td>18.8</td>
		</tr>
		<tr>
			<td>#6</td>
			<td>22.7</td>
		</tr>
		<tr>
			<td>#7</td>
			<td>19.1</td>
		</tr>
		<tr>
			<td>#8</td>
			<td>18.7</td>
		</tr>
		<tr>
			<td>#9</td>
			<td>15.0</td>
		</tr>
		<tr>
			<td>#10</td>
			<td>11.9</td>
		</tr>
	</tbody>
</table>
Table 1: MV Protein Yield from Peripheral Blood Samples. Shown is the amount of MVs per mL peripheral blood that was drawn from 10 donors. MV yields were quantified by Lowry assay.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>MVs/mL plasma [particle count]</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>6.42E+09</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>2.36E+10</td>
		</tr>
		<tr>
			<td>#3</td>
			<td>1.88E+09</td>
		</tr>
		<tr>
			<td>#4</td>
			<td>6.51E+09</td>
		</tr>
		<tr>
			<td>#5</td>
			<td>3.48E+09</td>
		</tr>
		<tr>
			<td>#6</td>
			<td>4.57E+09</td>
		</tr>
		<tr>
			<td>#7</td>
			<td>2.09E+09</td>
		</tr>
		<tr>
			<td>#8</td>
			<td>1.66E+09</td>
		</tr>
		<tr>
			<td>#9</td>
			<td>2.54E+09</td>
		</tr>
		<tr>
			<td>#10</td>
			<td>6.20E+09</td>
		</tr>
	</tbody>
</table>
Table 2: MV Particle Yield from Peripheral Blood Samples. MV were isolated from plasma samples of 10 donors and particle counts were determined by nanoparticle tracking analysis. Shown is the mean value from three independent measurements.

Watch this Scientific Journal Video about Isolation and Characterization of Microvesicles from Peripheral Blood at JoVE.com

Isolation and Characterization of Microvesicles from Peripheral Blood

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

isolation-and-characterization-of-microvesicles-from-peripheral-blood

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JoVE Journal

Biology

22.9K Views.  University Medical Center Göttingen. Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting. 

Video: Isolation and Characterization of Microvesicles from Peripheral Blood

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...