Name: fsl constructs: a simple method for modifying cell/virion surfaces with a range of biological markers without affecting their viability
Uploaded: 2011-08-05T15:00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability at JoVE.com

The authors thank Dan Bess from Sigma-Aldrich for presenting the video. We also thank Don Branch, Evgenii Cherny, Scott Chesla, Elizabeth Hadac, Amanda Harrison, Damien Heathcote, Annika Hult, Chuan-Ching Lan, Susannah McIntosh, Sarvani Komarraju, Elena Korchagina, Caroline Oliver, Martin Olsson, Stephen Parker, Igor Rodinov, Alexander Tuzikov, and Eleanor Williams for their results and contributions to the design, synthesis of FSL constructs and determining biological activity.

The following protocol describes the generic procedure for the insertion of FSL constructs (Figure 1) into biological membranes. For simplicity the protocol will only refer to cells (kodecytes), which are at a 100% concentration. However, the term cells is interchangeable with virions (kodevirions) or organisms or cellular structures and their concentration in diluent is not important, provided it is always consistent between tests, controls, and experiments. With red blood cells the packed cell volume is typically 80%, but with virions, embryos and other cells it is typically less than 1%. The method is very robust and will produce modified membranes when used by any of its variations, but refinement will be required to optimize and standardize the degree of modification required for specific applications.
1. Preparation of FSL Constructs
<ol>
	<li>To prepare the FSL construct stock solution first reconstitute the dry FSL product (Table 1) by the addition of 1.0mL of diluent (or as specified in the product insert) to the product vial. Briefly sonicate (30 seconds). This will prepare a 1mg/mL solution, which can be aliquoted into 100 &mu;L sterile containers and stored at 2-8&deg;C for up to one week, or frozen for up to 3 months.</li>
	<li>To prepare working FSL construct solutions for insertion, just prior to use briefly sonicate (30 seconds) the stock solution to homogenize any micelles. Dilute the FSL construct in buffer (preferably not containing lipids or highly hydrophobic material) to the concentration required or over a range if desired.</li>
</ol>
Notes:
<ol>
	<li>FSL constructs will usually insert into cells in lipid containing media but will typically require as much as a 50X higher FSL working concentrations than if in PBS or other lipid free media.</li>
	<li>The working range dilutions will depend on the application, the detection method sensitivity, the type of FSL construct, and the degree of modification required. Typically the range of dilution will be between 10-500 &mu;g of FSL per mL of diluent for carbohydrate FSL constructs and 1-100 &mu;g/mL for other FSL constructs such as fluorophores and biotin. </li>
	<li>If preparing insertion diluents containing multiple FSL construct, simply add the constructs together in the same diluent at their normal working concentration. If one construct is at much higher concentration than another, some adjustment (increase) in concentration may be required for the lesser construct. Alternatively constructs may be added sequentially, preferably with the highest concentration construct first, followed by the lesser.</li>
	<li>FSL construct concentrations above 1000 &mu;g/mL may introduce significant amounts of lipid into a cell membrane, and may change the shape of the cell or make it more susceptible to lysis.</li>
	<li>FSL construct working solutions should be stored at 2-8&deg;C and used within a few days.</li>
	<li>FSL constructs can be diluted in water but will have reduced stability and must be used within hours. If the package insert specifies water as the reconstitution diluent the product in the vial will also contain salts (as specified in the package insert).</li>
</ol>
2. Insertion of FSL Construct(s) into Membranes
<ol>
	<li>First wash the cells for FSL modification free of unbound lipids by centrifugation and using PBS or lipid free cell media as wash solution.</li>
	<li>Pack the cells or suspend in 100 &mu;L of diluent.</li>
	<li>Simultaneously prepare controls which ideally should be both unmodified cells (incubated with PBS rather than FSL solution) and/or cells modified with a benign but related FSL construct.</li>
	<li>Add 100 &mu;L of an appropriate dilution of FSL solution (containing 1 or more FSL constructs) to the cells and incubate for 1-2 hours at 37&deg;C. A similar result can be obtained by 6 hours incubation at 25&deg;C or overnight (18 hours) at 4&deg;C &#x2013; mixing is recommended every few hours if heavy cell suspensions are used.</li>
	<li>Wash (optional) twice with PBS or media to remove any free FSL constructs and prepare an appropriate suspension. </li>
</ol>
Notes:
<ol>
	<li>The diluent used to suspend the cells can be cell culture media, PBS, cell storage solutions, etc, but preferably without lipids ( e.g. fetal calf serum) or detergents (e.g. TWEEN). </li>
	<li>Different volumes (than 100 &mu;L) ratios (than 1:1 cells/FSL solution) or incubation conditions of time and temperature may be used provided the same ratio, concentration, and volume are used to obtain reproducible results. </li>
</ol>
3. Handling and Visualization
<ol>
	<li>Once the FSL modification process has been completed the kodecytes can be stored at 4-8&deg;C in lipid free media or used. </li>
	<li>Kodecytes and kodevirions generally behave the same as unmodified cells/virions and can be handled and viewed under normal testing systems (microscopy, serology, flow cytometry, etc). They usually have no special requirements except for the avoidance of solvents/detergents/lipids that may elute the lipid constructs from membranes.</li>
</ol>
Notes:
<ol>
	<li>Constructs will remain in the membrane of inactive cells, such as red cells, if stored in lipid free media for the life of the cell. In cells with active membranes the constructs will be internalized and metabolized, at a rate dependent on cell membrane activity. </li>
	<li>Dead cells will usually contain high levels of FSL constructs; this can be used to separate nonviable cells from viable cells. </li>
	<li>If kodecytes are stored (or used in vivo) in lipid containing media/environments, such as serum/plasma the construct will slowly elute from the membrane over a period of hours or days, depending on temperature and lipid concentrations/compositions. </li>
</ol>
4. Representative Results:
<ol>
	<li>The following representative results depend on the construct used (Table 1), its inserted concentration and the relative sensitivity and specificity of the detection system(s). In general all FSL constructs will insert into cells after 1 hour but the optimal conditions will need to be established for each situation.</li>
	<li>FSL constructs can be used to label the outside of a variety of live cells3-9,11,14. In the examples shown in Figure 2 embryos at different stages were used (as they are a fragile cell), but other cells (Figure 3) or enveloped virions (Figure 4) can also be labeled. FSL-Fluorescein allows for the direct labeling of surfaces (Figures 2-4), while FSL-biotin and other FSL constructs may require the use of a secondary detection system (usually avidin/antibodies/lectins) to show their presence (Figures 5).</li>
	<li>Blood group markers of human or animal specificity can be attached to cells, including red cells4-9 (Figure 6). As the amount of FSL construct on a kodecyte is controllable and reproducible4-6, kodecytes for antibody quantitation can be made (Table 2 and Figure 6). The actual source of cells is not important, they can be any species, or even of the same origin as the source of antibody, thereby ensuring on the only incompatible antigen is that introduced by the FSL7,8 (Figure 6).</li>
</ol>
<img src="/files/ftp_upload/3289/3289table2.jpg" alt="Table 2" /> 
Table 2. Representative serological reactions of three different FSL-A(tri) red cell kodecytes tested against dilutions of monoclonal anti-A. The 50 &mu;M solution of FSL-A(tri) when incubated with an equal volume of group O red cells produced strong A antigen positive cells, which could be detected by a 1:512 dilution of monoclonal anti-A. The 5- and 10 &#x2013;fold lower FSL-A(tri) solutions produced kodecytes with less blood group A antigen expression.
<img src="/files/ftp_upload/3289/3289fig1.jpg" alt="FSL construct overview" /> 
Figure 1. FSL construct overview. Like a flower FSL constructs consists of three major components. The functional head (F) in the case of the FSL construct can be a variety of different biologically functional groups, a spacer (S) designed to induce spacing of F away from the membrane, improve water dispersibility and be non-reactive with serum, while the diacyl lipid allows the construct to spontaneously incorporate into surfaces. (I) FSL-GB3 with a blood group Gb3 (or Pk) trisaccharide epitope Gal&alpha;4Gal&beta;4Glc&beta;. (II) FSL-A(tri) with a blood group A trisaccharide epitope GalNAc&alpha;3(Fuc&alpha;2)Gal&beta;. (III) FSL-Fluorescein. (IV) FSL-biotin with a single biotin F moiety. The functional group of structures in I-III are conjugated to an activated adipate derivative of dioleoylphosphatidylethanolamine (DOPE) while the spacer of structure IV is carboxymethylglycine-adipate based.
<img src="/files/ftp_upload/3289/3289fig2.jpg" alt="FSL-Fluorescein labeled murine embryos" /> 
Figure 2. FSL-Fluorescein labeled murine embryos. All images are of live embryos that were labeled when their zona pellucida (ZP) was intact (i.e. the FSL construct passed through the ZP to label the embryo inside). Images I-IV are of ZP-free embryos released from their ZP with acid tyrodes post labeling with FSL-Fluorescein. Identical staining occurs regardless of whether embryos were FSL modified with ZP intact or ZP free. Embryos are directly labeled with FSL-Fluorescein, by the 2 hour 37&deg;C method in serum free cell culture media, washed and then viewed under fluorescence microscopy. (I) ZP free two-cell murine embryo, also showing classic intense polar body staining. (II-III) ZP free four-cell and eight cell murine embryos, both showing shadowy staining of the cells which are outside the microscope plane of focus. (IV) ZP free 16 cell murine embryo. (V) ZP intact murine blastocyst embryos (d4-d5) where both the embryo and zona are labeled.
<img src="/files/ftp_upload/3289/3289fig3.jpg" alt="FSL-Fluorescein and zebrafish" /> 
Figure 3. FSL-Fluorescein and zebrafish14. (I) Microangiography on a 52 hpf (hours post fertilisation) zebrafish larvae directly injected into the circulation with FSL-Fluorescein. The zebrafish vasculature is stained. (II) FSL-Fluorescein heterogenous Zebrafish kidney tissue cells (ZK kodecytes) were created ex vivo then micro-injected into the circulation of a 52 hpf recipient Zebrafish. In vivo observations of the ZK kodecytes were made 2 hours post injection by imaging of the vasculature under fluorescence with time-lapse microscopy. Shown is a single video frame with large slow-moving or immobile cells (indicated with orange arrows) and fast moving cells (blurred images due to movement - indicated with green arrows). Labeling allows for real-time in vivo observation of the behavior and biodistribution the ZK kodecytes. (III) Oral uptake of FSL-Fluorescein achieved by immersing zebrafish embryos in FSL-Fluorescein containing media for up to 5 days. Washing was achieved by transferring embryos into media containing no FSL constructs (at least 6 hours is required but can be for several days). (IIIa) Bright field microscopy of the FSL-Fluorescein treated zebrafish corresponding to the adjacent fluorescence image (IIIb). The fluoresence was preferentially located in the intestinal tract. No staining was observed in untreated control embryos (IVa and IVb).
<img src="/files/ftp_upload/3289/3289fig4.jpg" alt="FSL-Fluorescein labeling of virions" /> 
Figure 4. FSL-Fluorescein labeling of virions10 VSV and H1N1. (I) Vesicular stomatitis virus (VSV) was directly labeled with 10 &mu;g/ml FSL-Fluorescein for 2 h at 37&deg;C, followed by fixing with 4% paraformaldehyde and then flow cytometry. No purification of the VSV kodevirion post FSL labeling was required. (II) Flow cytometry of swine testicular cells infected with human A/Puerto Rico/8/1934 (H1N1) kodevirions labeled using FSL-Fluorescein. Uninfected cells are seen as the black line while fusion of the H1N1 kodevirion with the ST cells results in a fluorescent cell (red line).
<img src="/files/ftp_upload/3289/3289fig5.jpg" alt="FSL-biotin labeled cells" /> 
Figure 5. FSL-biotin labeled cells and subsequent visualization via labeled avidin7,8. All cells were first labeled with FSL-biotin for 1 h at 37&deg;C, washed then reacted with fluorophore labeled avidin, washed and wet mounted for fluorescence microscopy. (I) Compiled confocal image of a murine embryo blastocyst. (II) Central confocal slice of the embryo from the preceding image. (III) Live motile human spermatozoa &#x2013; blurring occurs as a consequence of their motion. (IV) Fixed (4% paraformaldehyde post insertion) human spermatozoa. (V) Human erythrocyte. (VI) Fixed (4% paraformaldehyde pre-insertion) RL95 endometrial human carcinoma. (VII) Unfixed RL95 endometrial human carcinoma cell line. (VIII) Biotin RBC kodecytes observed in a blood sample taken 2 hours post intravenous infusion of kodecytes with (VIIIa) representing a field viewed under light microscopy while (VIIIb) is the same field viewed under fluorescence, identifying the two kodecytes present in the field-of-view. Calculating the ratio of kodecytes to unlabeled cells can be use as an indicator of survival. (IX) Live human endometrial biotin kodecytes visualized by binding to avidinylated beads.
<img src="/files/ftp_upload/3289/3289fig6.jpg" alt="FSL-Carbohydrate to add blood group markers for serologic profiling" /> 
Figure 6. FSL-Carbohydrate to add blood group markers for serologic profiling. (I) Human red cell Galili kodecytes were created with a set concentration of FSL-Galili (500 &mu;g/mL) and tested against dilutions of human serum. Human red cells do not naturally occur with the xenoantigen Galili antigen. Such kodecytes can be used to quantitative levels of antibody in serum. In this example the donor was determined to have an anti-Galili titre of 1:32. (II) By creating kodecytes with decreasing levels of FSL ("antigen titres"), an optimal antigen level to detect antibody can be determined (in this example Lea). Cells can be created to only give a positive result when the antibody level exceeds a specific titre. The level of FSL antigen required to give a positive result depends on the quality and level of antibody being detected. For carbohydrate antigens, a FSL solution of 100 &mu;g/mL will typically result in a strong positive reaction. (III) Human group O red cells were modified to have a specific level of A antigen (standardized A kodecytes), and used to accurately and reproducibly quantitate anti-A in human serum. In this example the anti-A titre in the group O serum tested is 1:32 and the kodecytes were prepared from the donor&#x2019;s own red cells. (IV) Blood group A and B antigens simultaneously inserted into a single group O red cell sample are used to create AweakBweak kodecytes. These kodecytes can be used for ABO quality control purposes. This result shows analysis of a specifically formulated AweakBweak kodecyte tested against anti-A and anti-B reagents and gives expected weak reactions.

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Immunol. 327, 1-21 (2009).</li><li>Henry, S., Bovin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+synthetic+peptidolipids,+glycolipids+and+other+lipid-linked+structures+to+create+designer+red+cells.">The development of synthetic peptidolipids, glycolipids and other lipid-linked structures to create designer red cells.</a> Transfusion. 48, 194A-194A (2008).</li><li>Frame, T., Carroll, T., Korchagina, E., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+glycolipid+modification+of+red+blood+cell+membranes.">Synthetic glycolipid modification of red blood cell membranes.</a> Transfusion. 47, 876-882 (2007).</li><li>Hult, A. K., Frame, T., Henry, S., Olsson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+evaluation+of+red+blood+cells+transformed+with+variable+amounts+of+synthetic+A+and+B+glycolipids.">Flow cytometry evaluation of red blood cells transformed with variable amounts of synthetic A and B glycolipids.</a> Vox Sang. 95, 180-180 (2008).</li><li>Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+red+blood+cells+for+laboratory+quality+control+use.">Modification of red blood cells for laboratory quality control use.</a> Curr. Opin. Hematol. 16, 467-472 (2009).</li><li>Oliver, C., Blake, D., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+neutralization+of+anti-A+and+successful+transfusion+of+A+antigen+incompatible+red+cells+in+an+animal+model.">In vivo neutralization of anti-A and successful transfusion of A antigen incompatible red cells in an animal model.</a> Transfusion. , (2011).</li><li>Oliver, C., Blake, D., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transfusion+reactions+and+predicting+in+vivo+cell+survival+with+kodecytes.">Modeling transfusion reactions and predicting in vivo cell survival with kodecytes.</a> Transfusion. , (2011).</li><li>Heathcote, D., Carroll, T., Wang, J. 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L., Brad Jones, R., Ramkumar, S., Sakac, D., Binnington, B., Henry, S., Lingwood, C. A., Branch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+globotriaosylceramide+analogue+inhibits+HIV-1+infection+in+vitro+by+two+mechanisms.">A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.</a> Glycobiology. 27, 515-524 (2010).</li><li>Barr, K., Diegel, O., Parker, S., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function-Spacer-Lipid+(FSL)+constructs+enable+inkjet+printing+of+blood+group+antigens.">Function-Spacer-Lipid (FSL) constructs enable inkjet printing of blood group antigens.</a> FEBS J. 277, 235-235 (2010).</li><li>Lan, C. -. 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Deborah A. Blank and Stephen M. Henry are employees and stockholders of KODE Biotech Limited the patent owner of KODE technology. Dan Bess is an employee of Sigma-Aldrich, which has sponsored the production of this video-article.

Modifying cells and virions with FSL constructs is a very simple and robust technique3-11. To describe and distinguish FSL construct modified cells and virions from unmodified cells, they are termed kodecytes and kodevirions respectively6-10, but only when the introduced functional group can be shown to be present on their membrane. When kodecytes are made with different concentrations of FSL constructs, they can be referred to by the concentration of the FSL construct solution used to create them, e.g. 15 &mu;g/mL A kodecytes, or if more than one FSL is used then a combined term, e.g. A+biotin kodecytes7,8. When different cells are to be compared, including the cell type in the description is recommended, e.g. red cell biotin kodecytes or endometrial biotin kodecytes7,8.
Although the process of insertion is very robust and insertion (albeit with different rates) will occur over a large temperature range from 4 - 37&deg;C and within minutes to hours, to obtain reproducible insertion strict control of contact time, temperature, FSL concentration (including diluent formulation), and to a lesser extent cell concentration are required. To date, all FSL constructs can be used to modify cells by the same technique4-11, however it is expected that certain conditions will be more favorable to work-flow and/or the optimal handling requirements of the cells/virions to be modified. The exact FSL concentration to achieve the desired effect needs to be determined by the user4. The resultant FSL construct modified cells/virions can usually be used in the same manner as an unmodified cell/virion in biological or analytical systems4-11. As the FSL constructs will slowly elute from modified surfaces when in contact with lipid solutions7,8, or be consumed by active cells11, these issues must be considered with respect to the level of signal or activity obtained over periods of time. Kodecytes can be fixed (e.g. glutaraldehyde/formalin) after insertion provided the fixative does not contain lipid-eluting solvents and the functional group is compatible with the fixative. Alternatively fixed cells can be modified with FSL constructs.
In addition to the modification of cell and virion surfaces FSL constructs can also be infused directly into the circulation of laboratory animals causing in vivo modification of circulating cells7 and to inhibit viruses12, toxins,12 and antibodies7. FLS constructs have also been used to decorate liposomes and can be printed onto paper surfaces13, where they immobilize, and can then be used in diagnostic assays.
The ability to easily modify live cells with an increasing range of FSL constructs should prove to be a useful research and development tool for the study of cell surface biology.

<table border="1">
	<tbody>
		<tr>
			<td>
				Sigma product number</td>
			<td>
				Sigma name</td>
			<td>
				Construct (KODE Biotech name)</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9307|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12346">F9307</a></td>
			<td>FSL-A(tri)</td>
			<td>FSL-A(GALNa3[Fa2]GALb)-SA1-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9557|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12347">F9557</a></td>
			<td>FSL-B(tri)</td>
			<td>FSL-B(GALa3[Fa2]GALb)-SA1-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9807|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12348">F9807</a></td>
			<td>FSL-GB3</td>
			<td>FSL-GB3(GALa4GALb4GLCb)-SA1-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9682|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12349">F9682</a></td>
			<td>FSL-Lea(tri)</td>
			<td>FSL-LEA(GALb3[Fa4]GLCNb)-SA1-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9182|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12350">F9182</a></td>
			<td>FSL-biotin</td>
			<td>FSL-CONJ(1Biotin)-SC2-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F9432|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12351">F9432</a></td>
			<td>FSL-Galili</td>
			<td>FSL-GALILI(GALa3GALb4GLCNb)-SA1-L1</td>
		</tr>
		<tr>
			<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F1058|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC&cm_sp=affiliate-_-Jove-_-12352">F1058</a></td>
			<td>FSL-Fluorescein</td>
			<td>FSL-FLRO4(fluorescein)-SA2-L1</td>
		</tr>
	</tbody>
</table> 
Table 1. Commercially available FSL constructs described in this paper.

fsl constructs: a simple method for modifying cell/virion surfaces with a range of biological markers without affecting their viability

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface1,2. However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required.
Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane. 
FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides3-12. FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions3-12.
The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37&deg;C4-10. During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes6-9, while virions are kodevirions10.
FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models7,8,10. All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety7,8,10,11.
The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions.

Watch this Scientific Journal Video about FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability at JoVE.com

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

Function-Spacer-Lipid (FSL) constructs allow the surface characteristics of living cells and virions to be modified without loss of vitality. The method requires only simple contact of an FSL construct solution with a cell/virion and spontaneous and stable surface incorporation occurs.

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

fsl-constructs-simple-method-for-modifying-cellvirion-surfaces-with

Research

JoVE Journal

Biology

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...