This work was supported by the NIH NHLBI R35HL155657 (N.M.) and the John C. Parker professorship (N.M.). We would like to thank Ms. Sierra J. Archibald for her helpful comments

The research was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol number: 14-2108).
1. Blood collection from donors
<ol>
	<li>Collect whole blood using clean venipuncture into the antecubital vein with a 21 G needle. Discard the first 3 mL of blood because this portion of blood may contain TF from perivascular cells.</li>
	<li>Draw 2.7 or 1.8 mL (depending on the size of the tubes) of blood into a vacutainer containing 3.2% sodium citrate (0.109 mol/L). Do not over- or underfill the tubes. Gently invert tubes immediately after blood collection to disperse the sodium citrate.</li>
	<li>Avoid agitation if the tubes are transported before processing. Prepare plasma within 2 h of blood collection.</li>
</ol>
2. Preparation of negative control plasma
NOTE: Samples should not be placed on ice at any time before the final freeze.
<ol>
	<li>Prepare negative control plasma using whole blood from healthy volunteers. Immediately after blood collection, prepare platelet-free plasma as follows.
	<ol>
		<li>Transfer blood samples from the vacutainer tubes to 15 mL tubes.</li>
		<li>Centrifuge blood samples at 2,500 &#215; g for 15 min at room temperature (20-24 &#176;C) without brake.</li>
		<li>Transfer the supernatant (platelet-poor plasma) to a new tube and repeat the spin under the same conditions.</li>
		<li>Transfer the supernatant (platelet-depleted plasma) to a new tube.</li>
		<li>Aliquot the sample into &#8805;100 &#181;L aliquots. Leave approximately 100 &#181;L at the bottom of the tube.</li>
		<li>Immediately freeze the platelet-depleted plasma at &#8722;80 &#176;C (Figure 1).</li>
	</ol>
	</li>
</ol>
3. Preparation of positive control plasma
NOTE: Response to lipopolysaccharide is variable between individuals.
<ol>
	<li>Transfer blood samples from the vacutainers to 15 mL tubes.</li>
	<li>Prepare positive control plasma using whole blood from healthy volunteers stimulated with lipopolysaccharide (LPS) from Escherichia coli O111:B4 (10 &#181;g/mL) for 5 h at 37 &#176;C with agitation.</li>
	<li>After 5 h incubation, follow the steps 2.1.2 to 2.1.6.</li>
</ol>
4. Isolation of extracellular vesicles from plasma
NOTE: EV pellets may not be visible. The defrosting time is dependent on sample volume but we usually defrost 100 &#181;L plasma for 30 min at 37 &#176;C.
<ol>
	<li>Defrost plasma samples at 37 &#176;C.</li>
	<li>Add 1 mL of HBSA without calcium [HBSA-Ca(-)] buffer [137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% (w/v) bovine serum albumin] to each 100 &#181;L of plasma sample in a 1.5 mL tube.</li>
	<li>Centrifuge plasma samples at 20,000 &#215; g for 15 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant down to 20 &#181;L without disturbing the EV pellet.</li>
	<li>Reconstitute the EV pellet by adding 1 mL of HBSA-Ca(-) buffer to each tube, pipette up and down at the location of the EV pellet, and vortex each tube before centrifuging for a second time at 20,000 &#215; g for 15 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant down to 20 &#181;L without disturbing the EV pellet.</li>
	<li>Reconstitute the EV pellet in 80 &#181;L of HBSA-Ca(-) and pipette up and down at the location of the EV pellet (Figure 2).</li>
</ol>
5. Option: Isolation of small extracellular vesicles using ultracentrifuge
<ol>
	<li>After step 4.3, collect the supernatant and ultracentrifuge at 100,000 &#215; g for 70 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant down to 20 &#181;L without disturbing the EV pellet.</li>
	<li>Reconstitute the EV pellet by adding 1 mL of HBSA-Ca(-) buffer to each tube and vortex each tube before ultracentrifuging for a second time at 100,000 &#215; g for 70 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant down to 20 &#181;L without disturbing the EV pellet.</li>
	<li>Reconstitute the EV pellet in 80 &#181;L of HBSA-Ca(-).</li>
</ol>
6. Measurement of extracellular vesicle tissue factor activity
NOTE: EV samples should be pipetted and vortexed well to mix before adding to wells. EDTA-disodium will inhibit the ability of FXa to cleave the chromogenic substrate. Therefore, EDTA-disodium cannot be used as a replacement for EDTA-tetrasodium in step 6.7.
<ol>
	<li>Add 40 &#181;L of an EV sample to two wells of a 96-well plate.</li>
	<li>Add 11 &#181;L of an inhibitory mouse anti-human TF IgG [(36.4 &#181;g/mL, final concentration 7.8 &#181;g/mL)] to one well of an EV sample and 11 &#181;L of control mouse IgG [(36.4 &#181;g/mL, final concentration 7.8 &#181;g/mL)] to the other well.</li>
	<li>Incubate the 96-well plate for 15 min at room temperature.</li>
	<li>During the incubation time, prepare 50 &#181;L/well of TF standards (0, 0.32, 0.63, 1.25, 2.5, 5, 10, and 20 pg/mL) in duplicate using relipidated recombinant TF.</li>
	<li>Prepare factor mix by mixing 4 mL of HBSA with calcium [HBSA-Ca(+)] [HBSA-Ca(-) + 10 mM CaCl2, final concentration 10 mM], 800 &#181;L of 900 nM FX in HBSA-Ca(+) (final concentration: 146.4 nM), and 120 &#181;L of 200 nM FVIIa in HBSA-CA(+) (final concentration: 4.8 nM).</li>
	<li>Add 50 &#181;L of the factor mix solution to each well, cover the plate with film, and incubate for 2 h in an incubator at 37 &#176;C.</li>
	<li>After 2 h, stop FXa generation by adding 25 &#181;L of HBSA ethylenediaminetetraacetic acid (EDTA) buffer [HBSA-Ca(-) + 25 mM EDTA-tetrasodium, final concentration 5 mM] and incubate for 5 min at room temperature.</li>
	<li>Reconstitute the chromogenic substrate that is cleaved by FXa to 4mM (add 8.7 mL of distilled water to a 25 mg vial).</li>
	<li>Add 25 &#181;L of the chromogenic substrate solution to each well, cover the plate with film and then with aluminum foil to protect it from light, and incubate for 15 min at 37 &#176;C.</li>
	<li>After 15 min of incubation, remove bubbles with a needle or by centrifugation at 1,500 &#215; g for 1 min.</li>
	<li>Read the plate at 405 nm using a plate reader with one mix before reading.</li>
	<li>Convert the FXa generation of each well to TF activity using the standard curve generated using relipidated recombinant TF.</li>
	<li>Calculate TF-dependent FXa generation (EVTF activity) using equation (1): 
	TF-dependent FXa generation (EVTF activity [pg/mL]) = Total FXa generation (control IgG well) - TF-independent FXa generation (anti-TF IgG well) (Figure 3) (1)</li>
</ol>

Improved Sensitivity for Tissue Factor Activity Assay in Extracellular Vesicles

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There are no competing interests to disclose.

Here, the protocol of our in-house EVTF activity assay is presented. There are three critical steps in the protocol. When reconstituting the EV pellet, it is important to pipette up and down at the location of the EV pellet even if it is not visible. Incomplete reconstitution of the EV pellet will result in a false negative or underestimation of EVTF activity values of the samples. Second, using HBSA-Ca(+) is critical in protocol step 6.5 because FXa generation cannot be generated without calcium. Third, using EDTA-tetrasodium but not EDTA-disodium to stop FXa generation is critical because the latter inhibits the ability of FXa to cleave the chromogenic substrate.
We recommend the following storage and use method for each reagent. We store assay buffers and the reconstituted substrate at 4 &#176;C and repeatedly use them until we run out of them. If we do not run out of the substrate within 2-3 weeks, we discard it because it turns yellow. We store recombinant TF, antibodies, FVIIa, and FX at -80 &#176;C and recommend making aliquots for one assay to avoid a freeze-thaw cycle.
In a preliminary study, we left plasma samples for 9 h at room temperature, refroze them at -80 &#176;C overnight, and performed the assay the next day. As a control, we thawed the same plasma and refroze it immediately. We found that the sample left for 9 h at room temperature had a 16% decrease in EVTF activity compared to the one that was thawed and refrozen immediately (Hisada and Mackman, unpublished data 2017).
In addition to the difference in response to LPS, the difference in the concentration of EVs in plasma from individual donors may explain the different EVTF activity of different donors. Indeed, we observed some variations in the concentrations of EVs in plasma from LPS-stimulated whole blood (1.9 &#177; 0.6 &#215; 1010 particles/mL, n = 6, mean &#177; standard deviation)30.
This in-house EVTF activity assay has several features that commercial assays do not have. First, the assay uses an anti-TF antibody to distinguish TF-dependent FXa generation from TF-independent FXa generation. Three commercial activity assays are claimed to measure TF activity in plasma but none of them uses anti-TF antibodies16. Therefore, these commercial assays simply measure total FXa generation but not TF-dependent FXa generation. Second, we use 2.4 nM of FVIIa (the final concentration in the reaction) to minimize TF-independent FXa generation by FVIIa. One commercial activity assay uses 12 nM of FVIIa in the reaction, which gives a high background22. Indeed, FVIIa activates FX&#160;linearly depending on FVIIa concentration in the absence of TF31. We summarized the studies that compared this in-house EVTF activity assay and commercial TF ELISA and an activity assay in Table 1.
Four response classifications of EVTF activity for citrated platelet-poor plasma are proposed: zero (0 to &#60;0.5 pg/mL); weak (0.5 to &#60;1.0 pg/mL), moderate (1.0 to &#60;2.0 pg/mL), and strong (&#8805;2.0 pg/mL)23. Notably, different anticoagulants affect the results of EVTF activity assays. For instance, higher levels of EVTF activity were observed using heparin after LPS stimulation compared to sodium citrate (Donor A: 4.6 (citrate) vs 28.6 (heparin) pg/mL and Donor B: 3.4 (citrate) vs 26.0 (heparin) pg/mL, respectively, Hisada and Mackman unpublished data 2017). In contrast, lower levels of EVTF activity were observed using EDTA after LPS stimulation compared to sodium citrate (Donor C: 3.9 (citrate) vs 1.4 (EDTA) pg/mL, Donor D: 3.8 (citrate) vs 0.4 (EDTA) pg/mL, respectively, Tatsumi and Mackman unpublished data 2016). These results indicate that EVTF activity data are not comparable when different anticoagulants are used.
There are a few limitations to the method. The coefficient of variation (CV) is relatively high compared to clinical assays. Indeed, the CV for the positive control in seven independent studies was 24%17,29. Isolation of EVs using centrifugation pellets not only EVs but also cellular debris. We previously analyzed the pellet of platelet-rich plasma by transmission electron microscopy and observed platelets, EVs, and cellular debris32. We use a fixed-angle rotor and have not experienced a swing-out rotor for 20,000 &#215; g centrifugation. We recently found that small EVs that can be pelleted with 100,000 &#215; g but not with 20,000 &#215; g have EVTF activity in patients with pancreatic cancer and COVID-1930. This indicates that this protocol does not measure EVTF activity from small EVs, such as exosomes. Notably, exosomes are sphingomyelin-rich EVs and the presence of TF-bearing exosomes has been reported30,33,34,35. We recommend pelleting EVs in plasma using 100,000 &#215; g to more accurately determine the total amount of EVTF activity in the sample. Of note, it requires an ultracentrifuge and a longer assay time because the centrifugation time increases from 15 min to 70 min.
This method can be applied to plasma samples from any kind of disease. EVTF activity is associated with disease severity and survival in patients with different diseases, including cancer, COVID-19, bacterial infection, and cirrhosis23,26,27.

Blood coagulation is initiated with the binding of factor (F) VII/VIIa to tissue factor (TF)1. The TF/FVIIa complex activates both FIX and FX to activate blood coagulation1. There are two forms of full-length, membrane-bound TF: encrypted and active. In addition, there is an alternatively spliced form of TF (asTF). Sphingomyelin and phosphatidylcholine in the outer leaflet of the cell membrane maintain TF in an encrypted state2,3,4. When cells are activated or damaged, phospholipid scramblase transfers phosphatidylserine and other negatively charged phospholipids to the outer leaflet1. The activation of cells also results in the translocation of acid sphingomyelinase to the outer leaflet where it degrades sphingomyelin to ceramide5. These two mechanisms convert encrypted TF to the active form. It is also proposed that protein disulfide isomerase mediates disulfide bond formation between Cys186 and Cys209 in encrypted TF, which results in the de-encryption of TF6,7,8. asTF is also present in the circulation but lacks the transmembrane domain and is therefore soluble9,10. Importantly, asTF has very low levels of procoagulant activity compared to full-length active TF10,11.
Extracellular vesicles (EVs) are released from resting, activated, and dying host cells, as well as cancer cells12. EVs express proteins from their parental cells12. Active TF-bearing EVs are released from activated monocytes, endothelial cells, and tumor cells into the circulation13,14,15. Levels of TF in plasma can be measured by activity- and antigen-based assays. Antigen-based assays include ELISA and flow cytometry16. There are two different activity-based assays: one and two-stage TF activity assays. The one-stage assay is based on a plasma-based clotting assay. The TF-containing sample is added to plasma and the time to form a clot is measured after re-calcification. The two-stage assay measures FXa generation of samples by adding FVII or FVIIa, FX, and calcium. FXa levels are determined using a substrate that is cleaved by FXa.
In both the one- and two-stage TF activity assays, the TF concentration is determined using a standard curve generated with recombinant TF. Two-stage assays have higher sensitivity and specificity than the one-stage assay. Many studies have confirmed that activity-based assays have higher sensitivity and specificity than antigen-based assays17,18,19,20,21. In addition, our in-house activity assay has higher sensitivity and specificity than a commercial activity assay22. Healthy individuals have very low or undetectable levels of EVTF activity in plasma. In contrast, individuals with pathologic conditions, such as cancer, cirrhosis, sepsis, and viral infection, have detectable levels of EVTF activity and this is associated with thrombosis, disseminated intravascular coagulation, disease severity, and mortality23,24,25,26,27,28. Here, we will describe this in-house two-stage EVTF activity assay.

<table><tbody><tr><td>1.5 mL tube for 20,000 x &lt;em&gt;g&lt;/em&gt; centrifuge</td><td>any company</td><td>N/A</td><td>We use the one from Fisher Scientific (Catalog number: 05-408-129).</td></tr><tr><td>1.5 mL tube for ultracentrifuge</td><td>any company</td><td>N/A</td><td>We use the one from Beckman Coulter (Catalog number: 357448)</td></tr><tr><td>15 mL tube</td><td>any company</td><td>N/A</td><td>We use the one from VWR (Catalog number: 89039-666)</td></tr><tr><td>21 G x .75 in. BD Vacutainer Safety-Lok Blood Collection Set with 12 in. tubing and luer adapter</td><td>BD</td><td>367281</td><td></td></tr><tr><td>96-well plate</td><td>any company</td><td>N/A</td><td>We use the one from Globe Scientific (Catalog number: 120338).</td></tr><tr><td>BD Vacutainer Citrate Tubes</td><td>BD</td><td>363083</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Centrifuge for 1.5 mL tube</td><td>any company</td><td>N/A</td><td>We use the Centrifuge 5417R (Eppendorf).</td></tr><tr><td>Centrifuge for 15 mL tube</td><td>any company</td><td>N/A</td><td>We use the Centrifuge 5810R (Eppendorf).</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate</td><td>Sigma Aldrich</td><td>E6511</td><td></td></tr><tr><td>Hepes</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>Human FVIIa</td><td>Enzyme Research Laboratory</td><td>HFVIIa</td><td>The solution should be diluted with HBSA-Ca(+).</td></tr><tr><td>Human FX</td><td>Enzyme Research Laboratory</td><td>HFX1010</td><td>The solution should be diluted with HBSA-Ca(+).</td></tr><tr><td>Inhibitory mouse anti-human tissue factor IgG, clone HTF-1</td><td>Fisher Scientific</td><td>550252</td><td></td></tr><tr><td>Lipopolysaccharide from Escherichia coli O111:B4</td><td>Sigma Aldrich</td><td>L2630</td><td>There are several lipopolysaccharide from different &lt;em&gt;E. coli.&lt;/em&gt; Different lipopolysaccharide have different potential to activate monocytes.</td></tr><tr><td>Mouse IgG</td><td>Sigma Aldrich</td><td>I5381</td><td></td></tr><tr><td>Pefachrome FXa 8595</td><td>Enzyme Research Laboratory</td><td>085-27</td><td></td></tr><tr><td>Plate reader</td><td>any company</td><td>N/A</td><td>We use the SpectraMax i3x from Molecular Devices</td></tr><tr><td>Re-lipidated recombinant tissue factor, Dade Innovin</td><td>Siemens</td><td>10873566</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima TLX</td><td></td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman Coulter</td><td>TLA-55</td><td></td></tr></tbody></table>

extracellular vesicle tissue factor activity assay

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX and FX. TF is released from cells into the circulation in the form of extracellular vesicles (EVs). The level of TF-positive (+) EVs is increased in various diseases, including cancer, bacterial and viral infections, and cirrhosis, and is associated with thrombosis, disseminated intravascular coagulation, disease severity, and mortality. There are two ways to measure TF+ EVs in plasma: antigen- and activity-based assays. Data indicates that activity-based assays have higher sensitivity and specificity than antigen-based assays. This paper describes our in-house EVTF activity assay based on a two-stage FXa generation assay. FVIIa, FX, and calcium are added to the TF+ EV-containing samples to generate FXa in the presence and absence of anti-TF antibody to distinguish TF-dependent FXa generation from TF-independent FXa generation. A chromogenic substrate cleaved by FXa is used to determine the FXa level, while a standard curve generated with a relipidated recombinant TF is used for the determination of the TF concentration. This in-house EVTF activity assay has higher sensitivity and specificity than a commercial TF activity assay.

A successful result gives a positive control value of &#8805;0.5 pg/mL and a negative control value of &#60;0.5 pg/mL. It is best to find a high LPS responder with &#62;1.0 pg/mL EVTF activity for a positive control. The representative result shows the EVTF activity of EVs isolated from plasma from the whole blood of 11 healthy donors, with and without LPS activation (Figure 4). Six out of eleven donors (Donors 2, 4, 5, 8, 10, 11) were medium to high LPS responders, whereas five out of eleven donors (Donors 1, 3, 6, 7, 9) were low LPS responders.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65840/65840fig01.jpg" /> 
Figure 1: Preparation of platelet-depleted plasma. The figure was modified from Hisada and Mackman29. Abbreviation: PDP = platelet-depleted plasma. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65840/65840fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65840/65840fig02v2.jpg" /> 
Figure 2: Isolation of extracellular vesicles from platelet-depleted plasma. The figure was modified from Hisada&#160;and Mackman29. Abbreviations: PDP = platelet-depleted plasma; EVs = extracellular vesicles. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65840/65840fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65840/65840fig03v4.jpg" /> 
Figure 3: Measurement of extracellular vesicle tissue factor activity. The figure was modified from Hisada and Mackman29. Abbreviations: TF = tissue factor; EVs = extracellular vesicles. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65840/65840fig03largev4.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65840/65840fig04.jpg" /> 
Figure 4: EVTF activity of plasma from whole blood of 11 healthy donors, with and without LPS activation. LPS activation was performed under the same condition as the positive control. The positive control was from a known LPS responder whereas the responses to LPS of 11 healthy donors were not known at the time of the experiment. White and black bars indicate with and without LPS activation, respectively. Abbreviations: EVTF = extracellular vesicle tissue factor; LPS = lipopolysaccharide. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65840/65840fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: A summary of the studies comparing this in-house EVTF activity assay, a commercial TF ELISA, and an activity assay. Abbreviations: EVTF = extracellular vesicle tissue factor; ELISA = enzyme-linked immunosorbent assay. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65840/65840_Table 1 (1).xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis at JoVE.com

Author Spotlight: High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis 

Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based assays.

Extracellular Vesicle Tissue Factor Activity Assay

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX ...

extracellular-vesicle-tissue-factor-activity-assay

Nanyang Technological University

Research

JoVE Journal

Medicine

698 Views.  University of North Carolina at Chapel Hill. Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based assays.

Video: Author Spotlight: High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis 

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cancer Research

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Immunology and Infection

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Assessing Blood Hypercoagulation of EV-Rich Plasma

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Author Spotlight: Deciphering Coagulation Disorders in Traumatic Brain Injury Patients 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...

Assessing Intracellular and Surface TF-Positive Platelets via Flow Cytometry

Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets

A subset of circulating human platelets stores Tissue Factor (TF) intracellularly, the key activator of the blood coagulation cascade and thrombus formation. Upon platelet activation, TF is exposed on the cell membrane, where it binds to FVII, ultimately leading to thrombin generation. Considering that (1) levels of TF-positive platelets increase in various clinical settings, contributing to the patient&#39;s prothrombotic phenotype, and (2) different drugs can modulate platelet-associated TF expression, a standardized method for assessing TF-positive platelets is valuable, as its evaluation has been controversial in the past. Here, we outline a protocol for measuring the percentage of TF-positive platelets using flow cytometry in whole blood and platelet-rich plasma (PRP)/washed platelets. This protocol aims to provide detailed instructions for quantifying the percentage of TF-positive platelets by assessing the protein (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. The first section provides essential information for correctly performing blood withdrawal to ensure that pre-analytical procedures do not affect the results. Next, the protocol focuses on sample preparation and labeling procedures for flow cytometry analysis. Detailed steps for cell stimulation, labeling, fixation, and permeabilization -- where necessary -- are outlined. Finally, instructions for flow cytometry settings to correctly identify the platelet population and analyze TF-positive events are described. Lastly, the method includes the procedure for preparing PRP if TF-positive platelets are to be measured in isolated platelets. Since only a subset of platelets contains TF, it is important to ensure that these platelets are not lost during the centrifugation steps required to obtain PRP.

This article outlines the protocol for quantifying the percentage of Tissue Factor (TF)-positive platelets using whole blood flow cytometry, assessing the protein: (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. Guidance is also provided for evaluating TF-positive platelets in platelet-rich plasma.