We thank J. Sedgewick for assistance in setting up the confocal microscope to capture images of silver grains; Supported in part by NIH research grants AI48484 (A.T.H.), AI20048 (R.A.), and AI066314 (C.J.M.); National Center for Research Resources grant. RR00169 (CNPRC, University of California, Davis).

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content/abstract/323/5922/1726">Li et al., Science 323, 1726-1729 (2009)</a>.
Combined in situ tetramer staining and in situ hybridization (ISTH) procedures
ISTH procedures can be divided into 4 steps: 1) IST staining of antigen-specific CD8+ T cells in tissues; 2) ISH to detect virus-infected-RNA+cells; 3) confocal microscopic imaging of IST-stained cells and viral-RNA+ cells; 4) image analysis, including construction of image montages and mapping of tetramer+ and virus RNA+ cells.
1. IST staining of antigen-specific CD8+ T cells in tissue sections.
 <ol>
 <li>Cut fresh tissues into 200 um thick sections using a vibratome or scalpel.</li>
 <li>Chill vibratome bath with sterile PBS-H and chill to 0-2&deg; C. Set the vibratome blade angle to a rather steep 27&deg;. Whenever possible, keep tissues chilled on ice in order to minimize degradation. Fresh tissue is also easier to section using a vibratome when it is chilled.</li>
 <li>Cut tissue with surgical scissors or scalpel into small approximately 0.5cm wide by 0.25 cm tall pieces. Put tissue pieces in a weigh boat or small dish and cover with ~40&deg;C melted 4% PBS buffered low melt agarose. Make sure tissue is in contact with the bottom of the dish. Push tissue pieces down if they float. Solidify on ice. This usually takes 3-5 minutes. </li>
 <li>Coat vibratome tissue block with Loctite glue. Cut agarose around tissue with a scalpel, and then using a forceps, carefully transfer the fragile agarose embedded tissue to a vibratome block coated in glue. Do not move the piece of tissue once it is set on the tissue block. Let dry approximately 10 minutes on ice.</li>
 <li>Mount tissue block in the vibratome and cut the tissue into 200um thick sections using a dead slow forward speed and relatively high amplitude. Speed and amplitude settings vary with each vibratome. If a vibratome is not available, or tissue is not amenable to vibratome sectioning, cut tissues into thin strips using a scalpel. </li>
 <li>Transfer sections with a paintbrush into a tissue chamber set in the well of a 24-well tissue culture plate containing 1 ml of chilled PBS-H. Put up to four tissue sections into each tissue chamber. Label the lid of the tissue culture plate with experimental sample information and secure lid to the plate with a rubber band. </li>
 <li>Alternatively, for tissues that don&#x2019;t cut well with vibratome, like gut, can cut as thin as possible strips with scalpel or razor blade. Put only one section per well for scalpel cut sections.</li>
 <li>Proceed to staining immediately after finished cutting tissues. Keep sections chilled to minimize degradation at all times until fixed. Do not let sections dry out. Keep sections in chilled sterile PBS-H.</li>
 <li>Incubate tissue sections over night with 0.5 mg/ml FITC conjugated tetramers. Can include mouse or non-rabbit antibodies directed at extracellular epitopes in this incubation, e.g. anti-CD8 antibodies, diluted 1:200 in blocking solution. Use 1 ml solution per well for this and all subsequent incubations, and perform this and all subsequent incubations at 4&deg;C with plates on a rocking platform. Keep tissue chambers containing different experimental samples separated by at least one empty well to prevent cross contamination of solutions in subsequent steps. </li>
 <li>After primary incubation, wash sections with chilled PBS-H twice (2X) for 20 minutes each. Do this by transferring the tissue chambers to a different 24-well tissue culture plate containing chilled PBS-H. Be careful not to drip contents from one experimental sample into another, when moving tissue chambers. For all subsequent incubations and washes similarly transfer the tissue chambers to a clean plate containing the appropriate solution. Monitor sections in tissue chambers during procedure to make sure that sections don&#x2019;t get stuck to the sides of the tissue chambers. </li>
 <li>Fix sections with fresh PBS buffered 4% paraformaldehyde for 2 hours at room temperature. Wash with cold PBS-H 2X 5 min each.</li>
 <li>Incubate with rabbit anti-FITC antibodies, e.g. Biodesign1:10,000, in blocking solution for one to three days. For co-labeling, can include non-rabbit antibodies directed at intracellular epitopes in this incubation as well. For this option, expose epitopes if needed and permeate and block the cells prior to the second incubation.</li>
 <li>If using antibodies directed at intracellular epitopes that require antigen retrieval from formaldehyde fixation, expose epitopes by boiling 3 times in 0.01M Urea, 10 seconds each time, in a microwave oven. Wait 2 minutes between each heating. Be very careful as boiling solution can force the sections out of the wells. If this happens, use a paint brush to push sections from the sides of the tissue chamber or from the lid of the plate back into the bottom of the appropriate tissue chamber. If antibodies do not require antigen retrieval omit this step.</li>
 <li>If using antibodies directed at intracellular epitopes, prior to the second incubation, permeabolize and block cells in tissue sections with PBS-H containing 0.3% triton X-100, and 2% normal goat serum for one hour. Then perform remaining antibody incubations in PBS-H containing 0.3% triton X-100 and 2% normal goat serum.</li>
 <li>After second incubation, wash sections in PBS-H for 20 minutes to several hours. Repeat twice for a total of three washes. Perform a final incubation with appropriate fluorescently labeled antibodies. For example, goat anti-rabbit-Cy3 and goat anti-mouse-Alexa 488 diluted 1:1000 each in blocking solution. Incubate for one to three days. Keep sections protected from light by wrapping the plates in tin foil during this incubation and thereafter, as light quenches fluorophores. </li>
 <li>Wash sections in PBS-H for 20 minutes to several hours. Repeat twice for a total of three washes. </li>
 <li>For IST combined with ISH post-fix sections post-fix in 4% paraformaldehyde for 1 hour to secure tetramers and antibodies in place, then rinse sections with PBS-H again and mount.</li>
 <li>Use a paintbrush to transfer sections to a microscope slide. Coat each section with glycerol/gelatin containing 4mg/ml n-propyl gallate or other mounting media containing a fluorophore preservative and cover with a coverslip. Store slides in light protected slide container at -20&deg;. Rinse tissue culture plates and remove labels on lids with alcohol. Can reuse plates.</li>
 <li>Analyze stained tissue sections with a confocal microscope. Then can proceed to ISH.</li>
</ol>
2. Detect virus-infected-RNA+ cells by in situ hybridization.
This in situ hybridization protocol was modified from our previously published protocols4,5 as follows:
<ol><li>Re-cut 5-8-micron-thick-sectionss from thick in situ tetramer stained sections.
<ol><li>200-micron-thick-sections are heated at 70&deg;C for 5 min to detach the section from the slide. </li>
 <li>The section is then embedded in OCT on dry ice. </li>
 <li>8-micron sections are cut with a cryostat from the thick sections and adhered to silanized slides.</li>
 <li>Slides can be stored at -80&deg;C in a sealed box for subsequent in situ hybridization.</li>
</ol>
</li>
<li>In situ Hybridization
<ol>
 <li>Rehydrate tissue sections by immersing slides for 3 min each in 90% ethanol and DEPC-treated water.</li>
 <li>Permeabilize tissue sections by heating in 10mM citrate buffer (pH 6.0) at 98&deg;C in a water bath for 15 min.</li>
 <li>Cool tissue sections at room temperature for 20-30 min. </li>
 <li>Wash twice in DEPC-treated water for 3 min.</li>
 <li>Acetylate tissue section by immersing slides in 0.25% acetic anhydride in 0.1 M TEA (triethanolamine, pH8.0) for 10 min.</li>
 <li>Transfer slides into 0.1 M TEA (pH8.0) for 5 min. </li>
 <li>Hybridize sections to 35S-labeled virus-specific (such as HIV-1, SIV) antisense probe or sense (negative control for HIV-1, SIV), or 35S-labeled LCMV riboprobes (pooled sense and antisense).</li>
 <li>Wash sections twice with 2xSSC for 5 min after overnight hybridization at 42&deg;C. </li>
 <li>Wash sections in STE (0.5 M NaCL, 1mM EDTA, 20 mM Tris-HCL, pH 7.5) for 5 min.</li>
 <li>Digest sections with RNases A and T1 in STE at 37&deg;C for 30 min</li>
 <li>Wash sections in 2x SSC plus 50% formamide for 5 min.</li>
 <li>Wash sections in 1x SSC 10 min.</li>
 <li>Wash sections in 0.5 x SSC 15 min. </li>
 <li>Dehydrate sections in 70, 80 and 100% ethanol containing 0.3 M ammonium acetate, each 5 min.</li>
 <li>Coat sections with nuclear track emulsion</li>
 <li>Dry in a dark room for 1 hour.</li>
 <li>Expose sections at 4&deg;C for 11 day (SIV) and 3 days (LCMV).</li>
 <li>Develop sections in D19 (Kodak, Cat# 146 4593) for 3 min, wash in dH2O 30 seconds, fix in Rapid Fixer (Kodak, Cat# 146 4106) for 4 min, wash in dH2O 5 min x 3 times.</li>
 <li>Dehydrate in 70%, 80% and absolute ethanol, 5 min each.</li>
 <li>Mount slides in fluorescent compatible Permount medium.</li>
</ol>
</li>
</ol>
3. Acquire confocal microscope images.
 <ol>
 <li>Capture ISTH images with a Bio-Rad MRC 1000 Confocal Microscope, or any other confocal microscope with Epi2-Blue Reflection device. </li>
 <li>Use Epi1-605 for the red tetramer fluorochrome, Epi2-522 DF32 for the green CD8 fluorochrome, and the Epi2-Blue Reflection for silver grains (ISH signal in the developed radioautographs) at 20x (for SIV) or 60x (for LCMV)</li>
 <li>Sequentially collect Z-serial images at 1-micron intervals in the three channels of each field. </li>
 <li>Acquire images from each section from left to right, and top to bottom, and name each image according to its row and column in an Excel File. </li>
 <li>Capture each image with a ~20% overlap with its neighboring images to avoid gaps in the reconstructed montage image. </li>
 </ol>
4. Image analyses: montage image reconstruction, cell centroid calculation and Spatial analysis.
<ol>
 <li>Project Z-serial images into a single image for each channel with Confocal Assistant (Version 4.02.). </li>
 <li>Automatically merge the projected image from three channels into one RGB image, using a Photoshop 7.0 Action procedure.</li>
 <li>Generate a montage image by stitching merged images together in layers in Photoshop 7.0. </li>
 <li>Associate individual silver grains and tetramer stain with cells, and assign the X,Y coordinates of the centers (centroids) of the SIV RNA+ and tetramer+ cells, using MetaMorph (version 7.1.3.) or Photoshop (available from the author) </li>
 <li>Export centroid data into Excel files as numeric numbers for each cell. E:T ratios are determined from the Excel files from the total numbers of tetramer+ and viral RNA+ cells in the section. </li>
</ol>
Spatial relationships between red tetramer+ and blue SIV RNA+ cells are captured by copying and pasting their respective plots from the Excel files into a Photoshop document. After decreasing the opacity of a layer to align and scale the grids so that they coincide, color-selecting and copying and pasting into a new layer the positions of the blue RNA+ cells, and then discarding the layer used to align the grids, the positions of the tetramer+ and viral RNA+ cells will be revealed in the flattened image.
5. Additional Notes
If cutting infectious tissue:
Work in a BSL2.5 lab. Take special precautions with razor blades. Put the razor into the vibratome blade mount with a forceps. If you can't put the blade in with a forceps, then put it in prior to adding tissue to the bath, and don't replace the blade. When finished cutting, remove the blade with a forceps. Spray blade with 70% EtOH or DMQ prior to removal. As always, dispose of blades in a sharps container. Change outer gloves or rinse in disinfectant after each contact with tissue or contaminated PBS in tank. When finished, if working with infectious material, add DMQ disinfectant to PBS in tank and let sit a couple of minutes prior to removing. Dispose of decontaminated PBS down the drain. Then rinse the tank with water to remove salt and disinfectant. Clean workspace and utensils with DMQ. Rinse utensils with water.
To Make tetramers from biotinylated MHC monomers:
Obtain biotinylated MHC class I molecules such as HLA-A *0201/B2m/peptide molecules produced with either Gag (SLYNTVATL), Pol (ILKEPVHGV), or irrelevant MART peptide (ELAGIGILTV) peptides from Beckman Coulter. Biotinylated HLA-A *0301/B2m/peptide molecules produced with either Gag (KIRLRPGGK) or Nef (QVPLRPMTYK) at the NIAID tetramer facility. Tetramers are generated by adding 6 aliquots of FITC labeled ExtraAvidin (Sigma) to biotinylated Mamu A*01/B2m/peptide monomers over the course of 8 hours to a final molar ratio of 4.5:1. The rationale behind adding the Extravidin slowly is that during the early time points there is an overabundance of monomer which assures that all of the Extravidin added gets all four biotin sites bound. Later in the reaction, this process is less efficient because there is less monomer left and more of a chance that ExtrAvidin will not have all 4 sites bound. If added all of the ExtrAvidin at once the reaction would be less efficient and more Extravidin molecules would have only 2 or 3 monomers attached.

<ol>
	<li>Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer-Williams, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274, 94-96 (1996).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+Edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting Edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> Journal of Immunology. 165, 617-61 (2000).</li><li>Qingsheng, L. i., Skinner, P. J., Ha, S. -. J., Duan, L., Mattila, T., Hage, A., White, C., Barber, D. L., O'mara, L., Southern, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen-specific+and+infected+cells+in+situ+predicts+outcome+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcome in early viral infection.</a> Science. 323, 1726-1729 (2008).</li><li>Haase, T., Haase, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perelson+Quantitative+Image+Analysis+of+HIV-1+Infection+in+Lymphoid+Tissue.">Perelson Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue.</a> Science. 274, 985-989 (1996).</li><li>Li, Q., Duan, L., Estes, J. D., Ma, Z. M., Rourke, T., Wang, Y., Reilly, C., Carlis, J., Miller, C. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peak+SIV+replication+in+resting+memory+CD4+T+cells+depletes+gut+lamina+propria+CD4+T+cells.">Peak SIV replication in resting memory CD4 T cells depletes gut lamina propria CD4 T cells.</a> Nature. 434, 1148-1152 (2005).</li><li>Li, Q. i. n. g. s. h. e. n. g., Estes, J. a. c. o. b. D., Schlievert, P. a. t. r. i. c. k. M., Duan, L. i. j. i. e., Brosnahan, A. m. a. n. d. a. J., Southern, P. e. t. e. r. J., Reilly, C. a. v. a. n. S., Peterson, M. a. r. n. i. e. L., Schultz-Darken, N. a. n. c. y., Brunner, K. e. v. i. n. G., Nephew, K. a. r. l. a. R., Pambuccian, S. t. e. f. a. n., Lifson, J. e. f. f. r. e. y. D., &amp;amp, J. o. h. n. V. C. a. r. l. i. s., Haase, A. s. h. l. e. y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+monolaurate+prevents+mucosal+SIV+transmission.">Glycerol monolaurate prevents mucosal SIV transmission.</a> Nature. , (2009).</li></ol>

The authors have nothing to disclose.

Since virus RNA is used as a marker for virus-infected cells in this report, all the reagents before hybridization should be RNAase free. The in situ hybridization procedure described here has been modified to preserve the fluorescent signal from in situ tetramer staining. Since the radioautographic signal from virus-infected-cells detected by in situ hybridization is at a depth of about one-cell-diameter, the thick sections used for in situ tetramer staining have to be recut into 5-8-micron-thick sections.
This novel technique of combining in situ tetramers and in situ hybridization (ISTH) described here enables simultaneous visualization, mapping and analysis of the spatial relationship of virus-specific CD8+ T cells and virus-infected target cells. This method can be applied to evaluate CD8+ T cell based vaccine and in other non-virus pathogen and host interactions. The image analysis method described here can also be adapted to study the spatial relationships of any two-cell populations interacting in situ, e.g. we recently used the centroid mapping procedure in studying of HIV-1 sexual transmission in the nonhuman primate model of simian immune deficiency virus infection of rhesus macaques6

Solutions and Reagents:
<ul>
 <li>PBS-H Phosphate buffered saline containing heparin (100ug/ml or 18.7U/ml) to inhibit RNASes</li>
 <li>RPMI tissue culture media containing heparin (100ug/ml or 18.7U/ml) to inhibit RNASes</li>
 <li>4% low melt agarose in PBS</li>
 <li>PBS with 2% normal goat serum (or serum from species in which the fluorophore conjugated antibodies were made).</li>
 <li>PBS with 2% normal goat serum and 0.3% triton X-100</li>
 <li>MHC class I tetramers conjugated to FITC</li>
 <li>Anti-FITC antibodies, e.g. BioDesign rabbit anti-FITC</li>
 <li>Fluorophore conjugated anti-rabbit IgG that has been highly cross adsorbed to other species IgG for use with multiple labeling, e.g. goat-anti-rabbit-Cy3</li>
 <li>Fluorophore conjugated anti-mouse IgG that has been highly cross adsorbed to other species IgG for use with multiple labeling</li>
 <li>Fresh PBS buffered 4% paraformaldehyde</li>
 <li>0.01M Urea</li>
 <li>Glycerol/gelatin containing 4mg/ml n-propyl gallate</li>
</ul>
Special equipment
<ul>
 <li>Vibratome</li>
 <li>Scalpel</li>
 <li>Razor blades</li>
 <li>Surgical scissors</li>
 <li>Loctite Quick Set Instant Adhesive (product # 46551)</li>
 <li>Forceps</li>
 <li>#2 camel hair paint brushes. Can trim with razor blade to desired thickness</li>
 <li>24-well flat bottomed tissue culture plates e.g. Falcon catalog #353226</li>
 <li>Tissue chambers</li>
 <li>Tin foil</li>
 <li>Cardboard slide folder e.g. Fisher catalog#12-587-10</li>
 <li>Confocal Microscope</li>
</ul>
IST Reagents Quick Reference
PBS-H (phosphate buffered saline with heparin)
<ul>
 <li>450 ml 1X PBS</li>
 <li>50 ml 1X PBS + 10X heparin</li>
</ul>
1x PBS + 10X heparin
<ul>
 <li>500 ml 1X PBS </li>
 <li>500 mg heparin powder (found on dry chemical storage shelf) </li>
</ul>
PBS-H/ 2% NGS (normal goat serum)
<ul>
 <li>49 ml PBS-H in Falcon tube </li>
 <li>1 ml NGS (found in 1 ml aliquots in -20 freezer) </li>
</ul>
PBS-H/ 2% NGS/ 0.3% Triton X-100
<ul>
 <li>49 ml PBS-H/ 0.3% Triton X-100 in Falcon tube </li>
 <li>1 ml NGS (found in 1 ml aliquots in -20 freezer) </li>
</ul>
PBS-H/ 0.3% Triton X-100
<ul>
 <li>500 ml PBS-H </li>
 <li>1.5 ml Triton X-100 (this is a thick liquid found on dry chemical storage shelves) </li>
</ul>
4% LMP Agarose
<ul>
 <li>2 g LMP agarose powder </li>
 <li>50 ml PBS </li>
 <li>Shake until mixed, microwave until powder dissolves. </li>
</ul>
GG-NPG (glycerol gelatin with n-propyl galate
<ul>
 <li>Place a bottle of glycerol gelatin in 50 degree water bath to melt</li>
 <li>Add 0.06 g of propyl galate (found on Terri&#8217;s shelf), shake well</li>
 <li>Keep in water bath, shaking occasionally until dissolved.</li>
 <li>Aliquot into 1 ml tubes (brown tubes)</li>
</ul>
4% paraformaldehyde
<ul>
 <li>4 g paraformaldehyde powder (found on dry chemical storage shelves) </li>
 <li>Put about 10 ml PBS-H in a graduated cylinder, add pf powder </li>
 <li>Add some water until volume reaches about 70 ml </li>
 <li>Add one dropper of NaOH. </li>
 <li>Stir until dissolved (usually about 20-30 minutes) </li>
 <li>Add HCl until pH reaches between 7-8. </li>
 <li>Bring volume to 100 ml with water. </li>
</ul>
Urea 0.01 M
<ul>
 <li>0.3 g urea (found on dry chemical storage shelves)</li>
	<li>500 ml water</li>
</ul>

a technique to simultaneously visualize virus-specific cd8+ t cells and virus-infected cells in situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

Watch this Scientific Journal Video about A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ at JoVE.com

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

a-technique-to-simultaneously-visualize-virus-specific-cd8-t-cells

Research

JoVE Journal

Biology

12.0K Views.  University of Minnesota. A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

Video: A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...