The authors are grateful to the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA) for providing normal brain samples. This study was supported by the National Institutes of Health (NIH) R01NS062787, the CJD Foundation, Alliance BioSecure, as well as the University Center on Aging and Health with the support of the McGregor Foundation and the President's Discretionary Fund (Case Western Reserve University).

1. Preparation of Brain Homogenate and Detergent -Soluble (S2) and -Insoluble (P2) Fractions
<ol>
	<li>Take 100 mg frozen human brain tissue and add 100 &mu;l lysis buffer (10 mM Tris, 150 mM NaCl, 0.5% Nonidet P-40 (NP-40), 0.5% EDTA, pH 7.4).</li>
	<li>Homogenize it on ice using pestle driven by a cordless motor, freeze it in dry ice for 5 min and homogenize it again using pestle driven by hands first and then by motor, and add 300 &mu;l or 800 &mu;l lysis buffer (this is either the 20% or 10% total brain homogenate, respectively).</li>
	<li>Centrifuge the 20% or 10% total brain homogenate at 1,000 x g for 10 min at 4 &deg;C to collect supernatant (S1) using a benchtop centrifuge.</li>
	<li>Ultracentrifuge 10% S1 at 35,000 rpm (100,000 x g) in an SW55 rotor (Beckman Coulter, Fullerton, CA) for 1 hr at 4 &deg;C. Transfer the supernatants (S2) that contain the detergent-soluble fraction into a clean tube. Resuspend the pellets that contain detergent-insoluble fraction (P2) in 100 &mu;l or 200 &mu;l the lysis buffer to make 10- or 5-fold concentrated preparation.</li>
	<li>For PK-digestion, incubate the sample with the same amounts of PK at 50 &mu;g/ml at 37 &deg;C for 1 hr for both S2 and P2 fractions. Add phenylmethylsulfonyl fluoride at the final concentration of 5 mM and equal volumes of SDS sample buffer (3% SDS, 2 mM EDTA, 4% &beta;-mercaptoethanol, 10% glycerol, 50 mM Tris, pH 6.8) to terminate the PK digestion. Boil the samples for 10 min and cool them at the room temperature for 5 min. They are ready for Western blotting.</li>
</ol>
2. Velocity Sedimentation in Sucrose Step Gradients
<ol>
	<li>Incubate 450 &mu;l 20% S1 with an equal volume of 2% sarkosyl in H2O for 30 min on ice.</li>
	<li>Add 0.5 ml each of 60%, 30%, 25%, 20%, 15%, and 10% sucrose into a Beckman tube with a maximal 5 ml capacity.</li>
	<li>Load 0.4 ml of S1 onto the top of 10-60% sucrose step gradients.</li>
	<li>Centrifuge at 48,000 rpm (200,000 x g, SW55 rotor) for 1 hr, at 4 &deg;C.</li>
	<li>Collect 283 &mu;l fraction each from top of the tube and obtain 12 fractions in total.</li>
	<li>Take 20 &mu;l of each fraction to a new Eppendorf tube, add 20 &mu;l sample buffer, and boil for 10 min.</li>
	<li>Cool samples for 4 min in hood, centrifuge at 1,000 x g for 1 min and vortex them. Load samples onto a pre-cast gel.</li>
</ol>
3. Size Exclusion Chromatography
<ol>
	<li>Use Superdex 200 HR beads (Pharmacia, Uppsala, Sweden) in a 1 x 30 cm column to determine the oligomeric state of PrP molecules.</li>
	<li>Seven molecular mass markers (Sigma, St. Louis, MI) including Dextran blue (2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), &beta;-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa) are loaded independently at the concentrations recommended by Sigma in 200 &mu;l sample volumes.</li>
	<li>The elution volume of blue dextran is used to determine the void volume (V0 = 8.45 ml) and the total volume (Vt = 24 ml) is provided by the product instruction. The peak elution volumes (Ve) are calculated from the chromatogram and fractional retentions. Kav, the partition coefficient (defining sample behavior), is calculated using the equation: Kav = (Ve - V0)/(Vt - V0). The calibration curve is determined by plotting the Kav of the protein standards against the log MW of the standards8.</li>
	<li>The molecular weight (MW) of the various PrP species recovered in different FPLC fractions is evaluated according to a calibration curve generated with the gel filtration of various standards.</li>
	<li>Inject 200 &mu;l S1 in lysis buffer containing 1% sarkosyl into the column for each run.</li>
	<li>Chromatography is performed in an FPLC system (GE Healthcare) at a flow rate of 0.25 ml/min and fractions of 0.25 ml each are collected using a fraction collector (Amersham Biosciences, RediFrac).</li>
	<li>Incubate 0.125 ml each fraction with 0.5 ml pre-chilled methanol at -20 &deg;C for 2 hr.</li>
	<li>Centrifuge samples at 13,000 x g for 30 min at 4 &deg;C in a benchtop microcentrifuge. Discard the supernatant and resuspend the pellet in 20 &mu;l SDS sample buffer as mentioned above, boiling for 10 min, cool to room temperature, load them onto a pre-cast gel.</li>
</ol>
4. Capture of PrP by g5p
<ol>
	<li>The g5p molecule (100 &mu;g) (kindly provided by Drs. Geoff Kneale and John McGeehan from the University of Portsmouth, UK) is conjugated to 7 x 108 tosyl activated magnetic beads by incubating 100 &mu;g g5p and 350 &mu;l g5p beads in 1 ml of phosphate-buffered saline (PBS) at 37 &deg;C for 20 hr. Attract g5p-beads to the sidewall of Eppendorf tubes by external magnetic force and then remove all solution. Wash the beads with 1 ml of PBS containing 0.1 % bovine serum albumin (BSA) for three times.</li>
	<li>Incubate the g5p-conjugated beads in 1 ml of 0.2 M Tris-HCl, pH 7.4 containing 0.1 % BSA at 37 &deg;C for 5 hr to block non-specific binding and then wash the beads with 1 ml of PBS containing 0.1% BSA for three times as mentioned above. Remove the solution and add 1 ml of PBS into the beads. The prepared g5p beads are stable for at least 3 months at 4 &deg;C.</li>
	<li>The capture of PrP by g5p is performed by incubating 100 &mu;l S1 fractions or P2 with 60 &mu;l g5p conjugated beads (10 &mu;g protein/6 X 107 beads) in 1 ml of binding buffer (3% Tween-20, 3% NP-40 in PBS, pH 7.5).</li>
	<li>After incubation with constant rotation for 3 hr at room temperature, the PrP-containing g5p beads are attracted to the sidewall of Eppendorf tubes by external magnetic force, allowing for easy removal of all unbound molecules in the solution.</li>
	<li>Following three washes in wash buffer (2% Tween-20 and 2% NP-40 in PBS, pH 7.5), g5p beads are collected and heated at 95 &deg;C for 5 min in the SDS sample buffer (3% SDS, 2 mM EDTA, 10% glycerol, 50 mM Tris-HCl, pH 6.8).</li>
	<li>After cooling samples for 5 min at room temperature, the samples are centrifuged at 1,000 x g for 5 min at room temperature. The supernatants are ready for Western blotting.</li>
</ol>
5. Western Blotting
<ol>
	<li>Load samples boiled in the SDS sample buffer onto 15% Tris-HCl Criterion pre-cast gels (Bio-Rad) at 150 V for ~80 min.</li>
	<li>The proteins on the gels are transferred to PVDF for 2 hr at 70V.</li>
	<li>After blocking membranes in 5% milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T), the membranes are incubated for 2 hr at room temperature with primary monoclonal or polyclonal antibody 3F4 (1:40,000), 1E4 (1:1,000), anti-N8 (1:6,000), or anti-C8 (1:6,000) for probing the PrP molecule.</li>
	<li>Following incubation with HRP-conjugated sheep anti-mouse IgG at 1:3000, or donkey anti-rabbit IgG at 1:3,000 the PrP bands are visualized on Kodak film using ECL Plus, in accordance with the manufacturer's protocol.</li>
</ol>
6. Representative Results
Compared to sporadic CJD samples, a small amount of iPrPC was detected in the P2 fraction in normal brains although most of PrPC was recovered in the S2 fraction (Figure 1). As indicated previously8, iPrP accounts for approximately 5-25% of total PrP including full-length and N-terminally truncated species.
Analyses using sucrose step gradient sedimentation revealed that while most of PrPC from non-CJD brains was recovered in the top fractions 1-3, small amounts of PrP were also detected in the bottom fractions 9-11 that normally contain large aggregates8 (Figure 2).
A variety of PrPSc species ranging from monomers, small oligomers to larger aggregates were isolated by gel filtration in the brain with Creutzfeldt-Jakob disease (Figure 3A). However, a small amount of larger aggregates with molecular weight greater than 2,000 kDa was also detected in insoluble fractions of normal brains (Figure 3C). Moreover, dimers and tetramers of PrPC were not only detected in insoluble fractions but also in soluble fractions (Figure 3B and 3C).
After PK and PNGase treatment, the PrP captured by g5p was detected with the 1E4 antibody against PrP97-105 8. Three PK-resistant core fragments termed PrP*20, PrP*19, and PrP*7 were detected, migrating at ~20 kDa, ~19 kDa and ~7 kDa, respectively (Figure 4, left panel). However, no PrP was detected when the 1E4 antibody was pre-incubated with a synthetic peptide that has a sequence identical to the 1E4 epitope (Figure 4, middle panel), indicating that bands detected by 1E4 are PrP fragments. Moreover, the anti-C antibody revealed two different PrP fragments migrating at ~18 kDa (PrP*18) and ~12-13 kDa (PrP-CTF12/13), in addition to PrP*20 (Figure 4, right panel).
<img alt="Figure 1" src="/files/ftp_upload/3788/3788fig1.jpg" /> 
 Figure 1. Detection of iPrPC and iPrPSc. After treatment with PNGase F at 1/10 of the total reaction volume at 37 &deg;C for 1 hr to remove glycans from the protein, full-length or N-terminally truncated PrP species in the soluble and insoluble fractions (S2 and P2) isolated by ultracentrifugation in brain samples from normal control (CTL) and sporadic CJD (sCJD) were detected with 3F4 against PrP106-112 (left panel), anti-N against PrP23-40 (middle panel), and anti-C against PrP220-231 (right panel). In CTL samples, a small amount of PrP is detected in P2, whereas a large amount is present in S2. In contrast, more PrP is detected in P2 than in S2 in sCJD samples.
<img alt="Figure 2" src="/files/ftp_upload/3788/3788fig2.jpg" /> 
 Figure 2. Sedimentation of PrP in sucrose step gradients. PrP in individual fractions from 1 to 11 of non-CJD brain sample S1 was detected by Western blotting with 3F4. Although most of PrPC was detected in top fractions 1-3, small amounts of PrP were also observed in bottom fractions 9-11. Moreover, the banding pattern of PrP from top and bottom is different: PrP recovered in the top fractions has a dominant upper band while PrP recovered in the bottom fractions has a dominant lower band. A PK-treated PrPSc was loaded as a control on the right side of blot.
<img alt="Figure 3" src="/files/ftp_upload/3788/3788fig3.jpg" /> 
 Figure 3. Detection of soluble and insoluble PrPC oligomers. Soluble and insoluble PrPC from normal human brains were separated by ultracentrifugation and then subjected to gel filtration, respectively. The molecular sizes of individual fractions were measured by running a group of molecular mass markers. (A) PrPSc species from sCJD brain samples. Two populations of PrP species were detected: gel filtration fractions 49 - 65 contain monomers and small oligomers, whereas fractions 27-33 contain large aggregates. The PrPC species from soluble fraction (S2) (B) and insoluble fraction (P2) (C) of normal controls were detected. PrP was probed with the 3F4 antibody. Dimers (fraction 55) and tetramers (fraction 51) of PrP were detected not only in P2 but also in S2 of normal brain samples (B and C). Large aggregates were only detected in P2 of normal samples (C).
<img alt="Figure 4" src="/files/ftp_upload/3788/3788fig4.jpg" /> 
 Figure 4. Detection of various PK-resistant iPrP fragments in g5p-enriched preparations from normal human brains. Samples enriched by g5p were treated with PK and PNGase F prior to Western blotting probing with 1E4 (left panel), 1E4 pre-incubated with a synthetic peptide that had a sequence identical to the 1E4 epitope (middle panel), and anti-C (right panel). 1E4 detected three PK-resistant PrP fragments termed PrP*20, PrP*19, and PrP*7 (left panel). After blocking of 1E4 with the peptide, no PrPres were detected (middle panel), indicating that the bands detected with 1E4 are PrP fragments. Anti-C revealed two addition PK-resistant PrP fragments termed PrP*18 and PrP-CTF12/13, in addition to PrP*20.

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	<li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions.+Proc.">Prions. Proc.</a> Natl. Acad. Sci. U.S.A. 95, 13363-13383 (1998).</li><li>Oesch, B., Westaway, D., W&#228;lchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., Prusiner, S. B., Weissmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cellular+gene+encodes+scrapie+PrP+27-30+protein.">A cellular gene encodes scrapie PrP 27-30 protein.</a> Cell. 40, 735-746 (1985).</li><li>Bolton, D. C., McKinley, M. P., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+purifies+with+the+scrapie+prion.">Identification of a protein that purifies with the scrapie prion.</a> Science. 218, 1309-1311 (1982).</li><li>Prusiner, S. B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F., Glenner, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+prions+aggregate+to+form+amyloid-like+birefringent+rods.">Scrapie prions aggregate to form amyloid-like birefringent rods.</a> Cell. 35, 349-358 (1983).</li><li>Hope, J., Morton, L. J., Farquhar, C. F., Multhaup, G., Beyreuther, K., Kimberlin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+polypeptide+of+scrapie-associated+fibrils+(SAF)+has+the+same+size,+charge+distribution+and+N-terminal+protein+sequence+as+predicted+for+the+normal+brain+protein+(PrP).">The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).</a> EMBO J. 5, 2591-2597 (1986).</li><li>Prusiner, S. B., Groth, D. F., Bolton, D. C., Kent, S. B., Hood, L. 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No conflicts of interest declared.

The combination of approaches reported here isolates consistently insoluble PrPC aggregates and soluble PrPC oligomers from the normal human brain. Ultracentrifugation at 100,000 x g for one hour is a classic method that has been widely used for the separation of the insoluble PrPSc from the soluble PrPC 14. While it is efficient, one of the cautions is to avoid contamination during pulling the supernatant (S2) after centrifugation. Since the gel profile of iPrPC is ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phenylmethylsulfonyl fluoride</td>
 <td>Sigma-Aldrich</td>
 <td>P7626</td>
 <td>72 mM in 2-propanol</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Sigma-Aldrich</td>
 <td>P2308</td>
 <td>2 mg/ml in H2O</td>
 </tr>
 <tr>
 <td>PNGase F</td>
 <td>New England Biolabs</td>
 <td>MWGF200</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge LE-80K, SW55 rotor </td>
 <td>Beckman Coulter</td>
 <td>Part No. 365668, 356860</td>
 <td></td>
 </tr>
 <tr>
 <td>Superdex 200 HR beads</td>
 <td>GE Healthcare</td>
 <td>17-1088-01</td>
 <td></td>
 </tr>
 <tr>
 <td>AKTA FPLC system</td>
 <td>GE Healthcare</td>
 <td>18-1900-26</td>
 <td></td>
 </tr>
 <tr>
 <td>Molecular weight markers</td>
 <td>Sigma-Aldrich</td>
 <td>MWGF200</td>
 <td>Varied </td>
 </tr>
 <tr>
 <td>Dynabeads M-280 magnetic beads</td>
 <td>Invitrogen</td>
 <td>143-01</td>
 <td></td>
 </tr>
 <tr>
 <td>3F4 antibody</td>
 <td>Covance</td>
 <td>SIG-39600</td>
 <td></td>
 </tr>
 <tr>
 <td>1E4 antibody</td>
 <td>Cell Sciences</td>
 <td>M1840</td>
 <td></td>
 </tr>
 <tr>
 <td>Ready gel 15% Tris-HCl pre-cast gels </td>
 <td>Bio-Rad</td>
 <td>345-0020</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of soluble and insoluble prp oligomers in the normal human brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

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10.7K ビュー.  Case Western Reserve University School of Medicine. 細胞プリオンタンパク質の新種（PRP C）は最近、ここで説明する方法を使用して、感染していない人間の脳で確認されています。そのうちのいくつかはまた、人間の脳から他のミスフォールドしたタンパク質凝集体を単離するのに有用であるが、これらの方法は、様々な種のPrPを単離することができる。

ビデオ: Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.