Dr. Pramod Nehete and Mrs. Bharti Nehete for macaque blood and PBMC samples; This work was partially supported by funds from NIH grant AI 46969, and all the cell culture media were produced by the Central Media Laboratory, which is funded by NIH grant CA 16672.

The procedures for the detailed phenotypic and functional analyses of total as well as memory subsets of T cells can be divided into four parts: 1) Cell preparation and antigenic stimulation, 2) Surface marker staining, 3) Intracellular cytokine staining and 4) Flow cytometry analyses.
1. Cell preparation and antigenic stimulation
<ol>
	<li>Both freshly isolated as well as cryo-preserved peripheral blood mononuclear cells (PBMC) were used in this protocol. The PBMC were isolated from heparinized or citrated venous blood samples of rhesus macaques by density gradient sedimentation using Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, ST. Louis, MO). Aliquots of PBMC were stored frozen in 90% FCS and 10% DMSO in liquid nitrogen.</li>
	<li>When using the cryo-preserved PBMC, the vials of frozen PBMC were removed from liquid nitrogen and rapidly thawed in a 37 C water bath, gently mixed, washed with RPMI-1640 (HyClone laboratories, logan, UT) to remove the freezing medium and re-suspended in complete media [CM; RPMI-1640 supplement with 10% heat-inactivated FCS (HyClone laboratories), 2mM L-glutamine (Sigma-Aldrich), 100U/ml penicillin/streptomycin (Invitrogen), and cultured in 6-well tissue culture plates overnight at 37&deg;C in a humidified 5% CO2 atmosphere. The next morning, viable cell counts were determined by the trypan-blue dye exclusion method and re-suspended in CM.</li>
	<li>The cells were treated differently for non-specific versus antigen-specific stimulation followed by cytokine analyses: (a) For non-specific stimulation with phorbol 12-myristate 13-acetate (PMA) and Ionomycin (I) (Sigma-Aldrich, St. Louis, MO), aliquots of cells in a volume of 0.1ml (1.0 x 106 cells/well) were plated in individual wells of 96-well tissue culture plates (BD Biosciences, Franklin lake, NJ). The PMA and Ionomycin were used at a final concentration of 50ng/ml and 500ng/ml, respectively. The final total volume for 96-well tissue culture plate is 0.2ml/well: (b) For antigen specific stimulation, 1.0 x 106 cells in a volume 1.0 ml were plated in individual wells of 24-well tissue culture plates (BD Biosciences, Franklin lake, NJ), where the wells were pre-treated as previously described with modification9. Briefly, the 24-well tissue culture plates were coated with 2.5 &mu;g/ml/well goat anti-mouse IgG (H + L) (Kierkegaard and Perry Laboratory, Gaithersburg, MD) in 50mM Tris solution (PH 8.6) for 4&deg;C overnight. The next morning the plates were washed twice with sterile PBS and co-stimulatory mAb CD49d, clone 9F10 (BD Biosciences; San Jose, CA) were added at 10 &mu;g/ml/well followed by incubating for one hour at 37&deg;C. After the incubation, the plates were washed twice with sterile PBS at room temperature. The antigens of interest (including cocktail of six HIV envelope peptides 8 were added at a final concentration of 10&mu;g/ml of each peptide. Additional wells with cells in CM alone were prepared as negative control. The final total volume for each well of the 24-well tissue culture plate was 1.0 ml.</li>
	<li>The cells were cultured for 6 hours at 37&deg;C in a humidified 5% CO2 atmosphere. Brefeldin A (Sigma-Aldrich, St. Louis, MO) was added to the culture at 10&mu;g/ml for the final 4.5 hours of stimulation. Subsequently, the cells were transferred to 5 ml polypropylene tubes and washed with cold (4&deg;C) flow wash buffer (Dulbecco's PBS (DPBS, Ca2/Mg2-free; Life technologies, Rockville, MD) with 2% heat-inactivated FCS), and then processed for staining with the various fluorescence labeled antibodies.</li>
</ol>
2. Immunofluorescence surface markers staining
<ol>
	<li>Stimulated cells were first stained with 1ul live/dead fixable aqua fluorescent reactive dye at 4&deg;C in the dark for 30 minutes, washed once with cold flow wash buffer.</li>
<li>Then, the following appropriately titrated fluorescence-labeled monoclonal antibodies were added to the cells: anti-CD3 PE-Cy7 (SP34-2), anti-CD8 Alexa700 (RPA-T8), anti-CD28 PerCP-cy5.5 (L293), anti-CD95 APC (DX2) and anti CD4 pacific blue (OKT4), all from BD Biosciences (Franklin lake, NJ), except CD4 pacific blue (OKT4) from eBioscience (San Diego, CA). The cells were incubated for 30 minutes at 4&deg;C in the dark.</li>
<li>For each experiment both compensation controls and fluorescence minus one (FMO) controls10,11 were utilized. Compensation controls were utilized to determine the spillover coeffecients of each individual stain into other detectors. For the compensation controls, a complete set of tubes containing cell suspensions from one of monkey were stained with fluorescent conjugated mAb individually as single color stains. FMO is a multicolor staining combination that contains all reagents but the one of interest and is used to determine the boundary between a positive and negative population by duplicating auto-fluorescence level and data present in fully stained sample.</li>
<li>Stained cells were then washed with cold flow wash buffer, and fixed in fixation/permeabilization solution (BD Biosciences; San Jose, CA) for at least 10 minutes (at this stage the cells can be stored overnight at 4&deg;C in the dark) before performing intracellular cytokine staining.</li>
</ol>
3. Intracellular cytokine staining
<ol>
	<li>Fixed cells were washed with flow wash buffer, and then Perm/Wash buffer (BD Biosciences; San Jose, CA) was added according to the instructions of the manufacturer. Briefly, cells were incubated in 0.1ml of the 1X Perm/Wash buffer for 10 minutes at 4&deg;C in the dark.</li>
	<li>Subsequently, the cells were incubated with the appropriately titrated FITC-labeled anti-IFN-&gamma; (B27) and PE-labeled anti-IL-2 (MQ1-17H12) for 60 minutes at 4&deg;C in the dark.</li>
	<li>Then the cells were washed two more times with the permeabilization solution</li>
<li>After the final wash, the cells were re-suspended in 1% paraformaldehyde in DPBS and subjected to Flow cytometry analysis within 24 hours.</li>
</ol>
4. Flow cytometry analysis
<ol>
	<li>The stained cells were acquired on a LSR II flow cytometer (BD Biosciences; San Jose, CA) or CyAn ADP (Dako, Carpinteria, CA). FACS data were analyzed by using FlowJo software (Tree Star, Ashland, OR).</li>
	<li>Figure. 1 shows the gating scheme utilized for the analyses of the different T cell subsets from a representative animal. The lymphocytes were first gated via forward scatter (FSC) versus side scatter (SSC), and then live lymphocytes were identified based on SSC and aqua-negative population. The T cells were then positively identified by CD3 expression followed by the detection of the CD4+ CD8- (CD4+ T cells) and CD4- CD8+ (CD8+ T cells) populations within the CD3+ T cell population. The CD4+ and CD8+ T cells were further distinguished into different subsets on the basis of CD28 and CD95 expression as na&iacute;ve (Tn, CD28+ CD95-), central memory (Tcm, CD28+ CD95+) and effector memory (Tem, CD28- CD95+) cells as described in the literature5, 10.</li>
	<li>Figure. 2 shows how the different subsets of CD4+ and CD8+ T cells were assessed for functional capacity in terms of cytokine production (INF-&gamma; and/or IL-2) in response to stimulation with PMA and ionomycin (PMA+I).</li>
</ol>
5. Representative FACS data
PBMC composition in rhesus macaques was determined using FACS analysis. Figure 1 shows the gating scheme utilized for the analyses of the different T cell subsets from a representative animal. The lymphocytes were first gated via forward scatter (FSC) versus side scatter (SSC), and then live lymphocytes were identified based on SSC and aqua-negative population. The T cells were then identified by CD3 expression. CD4+ CD8- (CD4+ T cells) and CD4- CD8+ (CD8+ T cells) populations within the CD3+ T cell population were also determined. As shown in Figure 2, the CD4+ and CD8+ T cells were further distinguished into different subsets on the basis of CD28 and CD95 expression as na&iacute;ve (Tn, CD28+ CD95-), central memory (Tcm, CD28+ CD95+) and effector memory (Tem, CD28- CD95+) cells. Figure 2A shows INF- &gamma; and IL-2 production in unstimulated and PMA / ionomycin stimulated CD4+ T cells, and subsets, and Figure 2B shows INF- &gamma; and IL-2 production in unstimulated and PMA / ionomycin stimulated CD8+ T cells, and subsets Note the distinct cytokine profiles elicited by stimulation in each subset.
<img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. Gating strategy used for flow cytometric analysis of PBMC composition in rhesus macqaues. Please <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="Figure 2a" border="0" /> <img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="Figure 2b" border="0" /> Figure 2. (A) Cytokine production profile of total CD4+ T cells and subsets. (B) Cytokine production profile of total CD8+ T cells and subsets. Please <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">click here</a> to see a larger version of 2a, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">or here</a> for a larger version of figure 2b.

<ol>
	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

Solution and Buffers
<ul>
 <li>Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO)</li>
 <li>RPMI-1640 medium (HyClone laboratories, Logan, UT)</li>
 <li>Fetal Bovine Serum (FBS), (HyClone laboratories, Logan, UT)</li>
 <li>L-glutamine (Sigma-Aldrich; St. Louis, MO)</li>
 <li>Penicillin/streptomycin (Invitrogen; Carlsbad, CA)</li>
 <li>Trypan blue reagent (Sigma-Aldrich; St. Louis, MO)</li>
 <li>50 mM Tris, PH 8.6; filtered and store at 4&deg;C</li>
 <li>Dulbecco's PBS (DPBS, Ca2/Mg2-free; Life technologies, Rockville, MD)</li>
 <li>Flow wash buffer: DPBS supplement with 2% FBS, store at 4&deg;C</li>
 <li>BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences; San Jose, CA)</li>
</ul>
Activation agents
<ul>
 <li>Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) </li>
 <li>Ionomycin (Sigma-Aldrich, St. Louis, MO)</li>
 <li>Costimulatory mAb CD49d, clone 9F10 (BD Biosciences; San Jose, CA) </li>
 <li>Affinity purified antibody F (ab )2 fragments of Goat Anti-Mouse IgG (H + L) (Kierkegaard and Perry Laboratory, Gaithersburg, MD)</li>
 <li>Specific activation agents could include peptide pools, single peptide, or whole protein</li>
</ul>
Cytokine secretion inhibitors
<ul>
 <li>Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO)</li>
 <li>Golgi Plug can be used alternatively (BD Biosciences; San Jose, CA)</li>
</ul>
Antibodies
<ul>
 <li>Live/dead fixable aqua fluorescent stain kit (Invitrogen; Carlsbad, CA)</li>
 <li>CD3 PE-Cy7 (SP34-2), (BD Biosciences; San Jose, CA)</li>
 <li>CD4 pacific blue (OKT4), (eBiosciences (San Diego, CA)</li>
 <li>CD8 Alexa700 (RPA-T8), (BD Biosciences; San Jose, CA) </li>
 <li>CD28 PerCP-cy5.5 (L293), (BD Biosciences; San Jose, CA)</li>
 <li>CD95 APC (DX2), (BD Biosciences; San Jose, CA)</li>
 <li>IFN- FITC (B27), (BD Biosciences; San Jose, CA)</li>
 <li>IL-2 PE (MQ1-17H12), (BD Biosciences; San Jose, CA)</li>
</ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

multicolor-flow-cytometry-analyses-cellular-immune-response-rhesus

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Immunology and Infection

21.0K Views.  MD Anderson Cancer Center - University of Texas. We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Video: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.