Name: in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues
Uploaded: 2017-09-22T15:00:00
Description: Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

This work was supported by Public Health Service grants from the National Institutes of Health (T32 DA007097, R01AI096966, andUM1AI26617).

1. Day 1: Fresh Tissue Sectioning and Primary Incubation
<ol>
	<li>Use a scalpel to cut fresh tissue into small (approximately 0.5 cm wide by 0.5 cm tall) pieces. Separately glue each tissue to a plunger and embed them with 3 - 5 mL of 4% low-melt agarose in PBS. Label the plunger with the tissue information using a sticker. Put it in a chilled holder in an ice bucket to solidify.</li>
	<li>Turn on the microtome and set the thickness of the sections to 200 &#181;m. Install a razor blade on the microtome and insert the ...

<ol>
	<li>Altman, J. D., et al. <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 (5284), 94-96 (1996).</li><li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+identify+peptide-specific+human+CD4(+)+T+cells+proliferating+in+response+to+influenza+A+antigen.">MHC Class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.</a> J Clin Invest. 104, R63-R67 (1999).</li><li>Steinert, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+memory+CD8+T+cells+reveals+regionalization+of+immunosurveillance.">Quantifying memory CD8 T cells reveals regionalization of immunosurveillance.</a> Cell. 161 (4), 737-749 (2015).</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> J Immunol. 165 (2), 613-617 (2000).</li><li>Haanen, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+virus-+and+tumor-specific+T-cell+immunity.">In situ detection of virus- and tumor-specific T-cell immunity.</a> Nat Med. 6 (9), 1056-1060 (2000).</li><li>Skinner, P. 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=In+situ+tetramer+staining+using+MHC+class+I+tetramers.">In situ tetramer staining using MHC class I tetramers.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. 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=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. 17, 4.1-4.9 (2004).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+HLA-DR*08032/E7+and+HLA-DR*0818/E7+tetramers+in+tracking+of+epitope-specific+CD4++T+cells+in+active+and+convalescent+tuberculosis+patients+compared+with+control+donors.">Use of HLA-DR*08032/E7 and HLA-DR*0818/E7 tetramers in tracking of epitope-specific CD4+ T cells in active and convalescent tuberculosis patients compared with control donors.</a> Immunobiology. 216, 947-960 (2011).</li><li>Bischof, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+autoreactive+CD4+T+cells+in+experimental+autoimmune+encephalomyelitis+after+primary+and+secondary+challenge+using+MHC+class+II+tetramers.">Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers.</a> J Immunol. 172, 2878-2884 (2004).</li><li>Massilamany, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+staining+with+major+histocompatibility+complex+class+II+dextramers+permits+detection+of+antigen-specific,+autoreactive+CD4+T+cells+in+situ.">Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ.</a> PLoS ONE. 9, e87519 (2014).</li><li>Massilamany, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+autoreactive+CD4+T+cells+in+brain+and+heart+using+major+histocompatibility+complex+class+II+dextramers.">In situ detection of autoreactive CD4 T cells in brain and heart using major histocompatibility complex class II dextramers.</a> J Vis Exp. (90), e51679 (2014).</li><li>Dileepan, T., Kim, H. O., Cleary, P. P., Skinner, 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=In+Situ+Peptide-MHC-II+Tetramer+Staining+of+Antigen-Specific+CD4++T+Cells+in+Tissues.">In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.</a> PLoS ONE. 10 (6), e0128862 (2015).</li><li>Tjernlund, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+Gag-specific+CD8++cells+in+the+GI+tract+of+SIV+infected+Rhesus+macaques.">In situ detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques.</a> Retrovirology. 7, 7-12 (2010).</li><li>Connick, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTL+fail+to+accumulate+at+sites+of+HIV-1+replication+in+lymphoid+tissue.">CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue.</a> J Immunol. 178, 6975-6983 (2007).</li><li>Hong, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Populations+of+CD8low/&#8722;+MHC+Class+I+Tetramer++SIV-Specific+T+Cells+in+Lymphoid+Follicles+and+Genital+Epithelium.">Localized Populations of CD8low/&#8722; MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium.</a> PLoS ONE. 4 (1), e4131 (2009).</li><li>Sasikala-Appukuttan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+and+dynamics+of+the+immunodominant+CD8+T+cell+response+to+SIV&#916;nef+immunization+and+SIVmac251+vaginal+challenge.">Location and dynamics of the immunodominant CD8 T cell response to SIV&#916;nef immunization and SIVmac251 vaginal challenge.</a> PLoS ONE. 8 (12), e81623 (2013).</li><li>Connick, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+simian+immunodeficiency+virus+replication+within+secondary+lymphoid+tissues+of+rhesus+macaques+is+linked+to+disease+stage+and+inversely+related+to+localization+of+virus-specific+CTL.">Compartmentalization of simian immunodeficiency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-specific CTL.</a> J Immunol. 193, 5613-5625 (2014).</li><li>Li, Q., et al. <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+outcomes+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection.</a> Science. 323, 1726-1729 (2009).</li><li>Li, Q., Skinner, P. J., Duan, L., 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=A+technique+to+simultaneously+visualize+virus-specific+CD8++T+cells+and+virus-infected+cells+in+situ.">A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ.</a> J Vis Exp. (30), e1561 (2009).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simian+immunodeficiency+virus-producing+cells+in+follicles+are+partially+suppressed+by+CD8++cells+in+vivo.">Simian immunodeficiency virus-producing cells in follicles are partially suppressed by CD8+ cells in vivo.</a> J Virol. 90, 11168-11180 (2016).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Lee, Y. J., Wang, H., Starrett, G. J., Phuong, V., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+specific+distribution+of+iNKT+cells+impacts+their+cytokine+response.">Tissue specific distribution of iNKT cells impacts their cytokine response.</a> Immunity. 43 (3), 566-578 (2015).</li><li>Abdelaal, H. M., Kim, H. O., Wagstaff, R., Sawahata, R., Southern, P. J., Skinner, 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=Comparison+of+Vibratome+and+Compresstome+sectioning+of+fresh+primate+lymphoid+and+genital+tissues+for+in+situ+MHC-tetramer+and+immunofluorescence+staining.">Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.</a> Biol Proced Online. 17, (2015).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</li></ol>

IST combined with IHC provides an essential tool for detecting, characterizing, and quantifying antigen-specific CD8 T cells in native environments with the context of other cells and tissue structures. Here, we described detailed procedures for IST combined with IHC, followed by quantitative image analysis, to determine the location, abundance, and phenotype of antigen-specific CD8 T cells in lymph nodes from rhesus macaques. Similar staining can be applied to human, mouse, or other species tissues for which MHC-I tetra...

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of peptide/MHC class I tetramer staining of antigen-specific CD8 T cells1 and the more recent development of MHC class II tetramer staining of CD4 T cells2 by flow cytometry revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry does not allow for the detection of the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues3.
We and others have developed methods using peptide-loaded MHC class I and class II tetramer or multimer reagents to stain antigen-specific CD8 and CD4 T cells in tissues4,5,6,7,8,9,10,11,12,13. These IST methods allow for the determination of the location, abundance, and phenotype of antigen-specific CD8 and CD4 T cells in tissues and provide a means to detect of these cells relative to other cells and structures in the tissues. Our group has extensively used MHC-I tetramer staining to study human immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-specific CD8 T cells in lymphoid, genital, and rectal tissues to gain an understanding of HIV and SIV immunopathogenesis and to identify correlates of successful vaccination strategies14,15,16,17. In addition, we also developed a technique that combines IST with in situ hybridization (ISH) to localize and quantify virus-specific CD8 T cells and virus-infected cells in tissues and to determine the in vivo effector-to-target levels18,19.
Here, we describe a protocol using peptide-loaded MHC-I tetramers to stain antigen-specific CD8 T cells in fresh tissue sections, to counterstain tissues using IHC, and to quantify cells with specific phenotypes in specific tissue compartments. These procedures are the same as were used in our recent publication by Li et al., in which we determined the location, abundance, and phenotype of SIV-specific T cells in lymphoid tissue during chronic SIV infection in macaques20.
For this procedure, fresh tissues are sectioned and incubated overnight with peptide-loaded MHC-I tetramers conjugated to fluorescein thiocyanate molecules (FITC). They are then fixed in paraformaldehyde. After fixing the tissue, the signal from the MHC tetramers is amplified using rabbit anti-FITC antibodies and incubated with fluorescently tagged anti-rabbit IgG antibodies, which further amplify the signal from the bound tetramers. IHC is used in conjunction with IST to characterize antigen-specific T cells and surrounding cells. Antibodies that recognize epitopes on the surface of cells or in the extracellular space are included in the primary incubation with the tetramers. Antibodies that recognize intracellular epitopes require permeation of the cell wall prior to staining. The stained tissue sections are imaged using a confocal microscope and analyzed using confocal software. Labeled cells are quantified using confocal microscopy software or ImageJ. The described protocol can be used to stain essentially any antigen-specific CD8 T cell in any tissue for which MHC-I tetramers are available.

<table align="center" border="1" cellpadding="0" cellspacing="0" style="width:906px;" width="906">
	<tbody>
		<tr>
			<td>MHC-I monomer</td>
			<td>NIH tetramer core facility</td>
			<td></td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>ExtrAvidin-FITC</td>
			<td>Sigma-Aldrich</td>
			<td>E2716</td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBL6391V</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>J.T.Baker</td>
			<td>4204-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol/gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBH2672V</td>
			<td></td>
		</tr>
		<tr>
			<td>n-propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>rat-a-h-CD8 (1:500)</td>
			<td>Acris</td>
			<td>0714</td>
			<td>Antibody unstable, use single use frozen aliquot</td>
		</tr>
		<tr>
			<td>m-a-h-CD20 (1:500)</td>
			<td>NOVOCASTRA</td>
			<td>6026819</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-h-Ki67 (1:500)</td>
			<td>Vector</td>
			<td>6022201</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-m-A488 (1:2000)</td>
			<td>Jackson Immunoresearch</td>
			<td>124083</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rb-Cy3 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>106232</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rat-Cy5 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>118088</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-h-IgM-Dylight649 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>86579</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresstome: VF-300 Microtome</td>
			<td>Precisionary Instruments, LLC</td>
			<td>1079</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Set Instant Adhesive</td>
			<td>Loctite</td>
			<td>46551</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well flat bottomed tissue culture plates</td>
			<td>Falcon</td>
			<td>353226</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Globe scienfitic Inc.</td>
			<td>#1321</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor&#160; blade</td>
			<td>Ted Pella, Inc</td>
			<td>121-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Disposable Scalpel</td>
			<td>FEATHER SAFETY RAZOR CO. LTD.</td>
			<td>No. 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Round paintbrush #2</td>
			<td>PRINCETON ART &#38; BRUSH CO.</td>
			<td>4350R</td>
			<td>Can trim as needed with razor</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW_Viewer4.0</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

Figure 1 shows how to collect confocal images using a confocal microscope. Figure 2 demonstrates quantitative image analysis using ImageJ. Figures 3 and 4 show representative images of lymph node tissues from an SIV infected rhesus macaque stained with MHC tetramers, CD8 antibodies, and CD20 antibodies, and serve to demonstrate the specificity of the MHC-tetramer staining. Fi...

Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

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