We thank Rebecca Nishi, M.S., and the technicians of the Christopher &amp; Dana Reeve Foundation Animal Core Facility at UC Irvine for their help in animal surgery. We also thank Gabriella Funes, Usha Nekanti and Denisse Moreno for technical assistance, manuscript and video shoot preparations and animation. This research was supported by NINDS (RO1 NS43428-01 to AJA), the Paralysis Project of America (PPA-32574 to HXN), and California Institute for Regenerative Medicine (CIRM) Stem Cell Training Award T1-00008 to HXN. KDB was supported by the training program in molecular and cellular neuroscience at UC Irvine (T32 NS007444).

1. Dissociation of spinal cord tissue
<ol>
<li>Spinal cord segments T8-T10 of non-injured or contusion spinal cord injured (injury at T9) Sprague Dawley rats were dissected and mechanically dissociated with fine scissors in HBSS at room temperature, as previously described 1. Prior to tissue dissociation, whole spinal cord columns were kept on dry ice for 5 minutes before the extraction of cord segments T8-T10.</li>
<li>Tissue bits were retrieved by centrifugation (1 minute, 1000 rpm, room temperature) and enzymatically dissociated with 2.5 mg trypsin and 5 mg collagenase in 5 ml DME (Dulbecco&#x2019;s Modified Eagle&#x2019;s Media) for 20 minutes at 37&deg;C before trituration (&tilde;10 times at room temperature) with a glass Pasteur pipette (9-inches).</li> 
<li>10 ml of DME + 10% fetal bovine serum was added to cells to inhibit enzymatic activities and then was filtered through a 40 &mu;m cell strainer. After a quick spin, the cell pellet was resuspended in 6 ml of HBSS and overlayed on OptiPrep gradient solutions described below.</li>
</ol>
2. Creating OptiPrep gradient solutions
<ol>
<li>Diluted OptiPrep was constructed by diluting OptiPrep 1:1 with MOPS Buffer (0.15 M NaCl, 10 mM MOPS, pH 7.4).</li>
<li>Four OptiPrep gradient solutions were made by mixing 350, 250, 200, or 150 &mu;l diluted OptiPrep with HBSS to a final volume of 1 ml (Table 1).</li>
<li>The four solutions were slowly and carefully placed in layers in a 15 ml conical tube with the least dilute at the bottom and the most dilute on the top (Figure 1A).</li>
</ol>
3. Separating lipid/myelin debris from cells
<ol>
<li>6 ml of dissociated spinal cells in HBSS was carefully layered on top of the OptiPrep gradient solutions.</li>
<li>Tube containing cells and gradient solutions was centrifuged (15 minutes, 1900 rpm or 726 RCF, 20 &deg;C) using an Eppendorf Centrifuge 5810R with a swing-bucket rotor (A-4-62), separating the cell solution into distinct layers with lipid/myelin debris (top 7 ml of tube), followed by 3 layers of neurons, with inflammatory cells, glia, and red blood cells in the pellet. Although most inflammatory cells, including monocytes, macrophages, PMN granulocytes, and lymphocytes were found in the pellet, some large sized-activated macrophages could be found in layers above the pellet.</li>
<li>The lipid/myelin debris layer (top 7 ml) was carefully aspirated and removed. Cells were then washed and resuspended in 2.5 ml HBSS and used for immunolabeling below.</li>
</ol>
4. Immunolabeling of specific immune cells for flow cytometry
<ol>
<li>Cells (500 &mu;l) collected from spinal cord preparations were pelleted and resuspended in 0.85% ammonium chloride (diluted in distilled water) for 5 minutes to lyse red blood cells.</li>
<li>Cells were washed with 500 &mu;l HBSS and then blocked for 30 minutes in normal rabbit or mouse serum at room temperature.</li>
<li>Cells were washed and incubated at room temperature for 1 hour with antibody (Rabbit anti-PMN FITC, Accurate Chemical and Scientific; Mouse anti-rat ED1 Alexa 488, Serotec; or Mouse anti-rat CD3 Alexa 488) or isotype IgG solution diluted in HBSS (all at 1:100 dilution), as described previously 1.</li>
<li>Cells were washed twice after each step above and then resuspended in 300 &mu;l HBSS after the final step.</li>
</ol>
5. Cell quantitative assessment by flow cytometry
<ol>
<li>Samples were analyzed on a FACS Calibur (Becton-Dickinson) flow cytometer using Cell Quest software. 5,000 events per sample were read for all samples, and data analysis was completed with Summit (DakoCytomation).</li> 
<li>Flow cytometric gates were set using control IgG isotype labelled cells or spinal cord cells from uninjured control animals to set baseline values for normalization across time points as described previously 1. The mean values of positively labelled cells were expressed as percent (&plusmn; S.E.M.) of the entire sample.</li>
</ol>
6. Representative Results: 
The ability of an OptiPrep gradient to remove myelin debris and improve immune cell detection by flow cytometry was assessed by quantifying spinal PMNs at 1 dpi (Figure 1B), the previously reported peak of PMN infiltration after SCI 1-3. Alternatively, identically dissected spinal cords were enzymatically dissociated without OptiPrep-removal of myelin debris and were similarly assessed in parallel for PMN infiltration by flow cytometry. As we have reported previously 1, there was a shift in the position of events in samples isolated with OptiPrep gradient purification method compared to enzymatic dissociation alone, demonstrating the percentage of PMNs detected in samples with intact myelin debris was only a fraction (0.5%) of the percentage of PMNs detected (5.1%) in OptiPrep purified samples (Figure 1B). These data suggest that myelin debris in tissue preparations can obscure flow cytometric readings, and demonstrate that removal of myelin debris enhances the sensitivity of immune cell detection in the injured spinal cord tissue.
Having established the sensitivity of the OptiPrep gradient system for cell detection in the injured spinal cord, we characterized changes in cellular infiltration over a period ranging from 2 hrs to 180 dpi and established a novel multiphasic response of cellular inflammation after SCI that included PMNs, macrophages/microglia and T-cells. As confirmed by previous studies 1-3, PMNs first entered the cord at 2 hrs post-injury and peaked at 1 dpi (Figure 2 &amp; 4). Macrophages/microglia on the other hand 1,4,5, were not detected in the injured spinal cord until 3 dpi, and peaked initially at 7 dpi (Figure 3 &amp; 4). Surprisingly, we show for the first time that macrophages/microglia peaked for the second time 60 dpi and remained elevated through 180 dpi (Figure 3 &amp; 4). Similar to macrophages/microglia, T-cell infiltration was predicted to peak 7 dpi 5-7; however, flow cytometry did not detect any changes in T-cell number in the first 7 dpi, though an elevated number of T-cells was detected at 9 dpi (1.6%) (Figure 4). While T-cell number was decreasing by 10 dpi, a persistent T-cell response was observed through 180 dpi, at which time 4.4% of cells were labelled for CD3 (Figure 4). 
Together, these data demonstrate a time-dependent multiphasic response of cellular inflammation after SCI (Figure 4); the initial phases of cellular inflammation were comprised of the early peak of PMNs 1 dpi followed by a peak of ED1+ macrophages/microglia 7 dpi and T-cells 9 dpi, while the later phases were composed of all three cellular populations rising after 14 dpi and persisting through 180 dpi, with a notable second peak of macrophages/microglia at 60 dpi. This timecourse study and the quantitative data generated by flow cytometry have been validated previously, showing comparable results to data collected from quantitative stereology of immunolabeled spinal cord sections 1. 
<img src="/files/ftp_upload/2698/2698table1.jpg" alt="Table 1"/> 
Table 1. Preparation of OptiPrep gradient for the removal of myelin debris. Four OptiPrep gradient solutions are made using HBSS and diluted OptiPrep (1:1 dilution of OptiPrep and MOPS).
<img src="/files/ftp_upload/2698/2698fig1.jpg" alt="Figure 1"/> Figure 1. OptiPrep gradient densities separate most myelin/debris from injured spinal cord tissues/cells and improve immune cell assessment by flow cytometry. (A) Myelin/debris was separated from cells (neurons, glia and immune cells) after centrifugation of dissociated spinal cord tissue through an OptiPrep gradient followed by aspiration of myelin/debris layer. (B) PMN number in the injured spinal cord, showing increased sensitivity in detecting PMNs after debris removal (Student&#x2019;s t-test, p = 0.0001). All flow cytometric gates were set using labeled cells from uninjured animals; n=5 per group, mean &plusmn; SEM. 5,000 events per sample were read for all samples and the mean values of positively labeled cells were expressed as percent (&plusmn; S.E.M.) of the entire sample.
<img src="/files/ftp_upload/2698/2698fig2.jpg" alt="Figure 2"/> Figure 2. Detection of PMN infiltration in the injured spinal cord by flow cytometry. After a moderate (200 kd) contusion injury at T9, PMNs quickly entered the spinal cord starting 2 hrs (B) post-injury and peaking 1 dpi (C). All flow cytometric gates were set using labeled cells from uninjured animals (A).
<img src="/files/ftp_upload/2698/2698fig3.jpg" alt="Figure 3"/> Figure 3. Detection of macrophages/microglia infiltration in the injured spinal cord by flow cytometry. After a moderate (200 kd) contusion injury at T9, ED1+ macrophage/microglial number increased in the injured spinal cord starting at 3 dpi (D), peaked acutely at 7 dpi (E), dropped to low levels at 14 dpi (F), before rising to a second peak at 60 dpi (G), and remained in the injured spinal cord up to 180 dpi (H). IgG isotype control labeling of 7 dpi spinal cells (B) showed minimal antibody-labeling background and no difference to antibody-labeled cells from 2 hrs post-injured (C) or uninjured (A) animals. All flow cytometric gates were set using labeled cells from uninjured animals.
<img src="/files/ftp_upload/2698/2698table2.jpg" alt="Table 2"/> 
Table 2. Animal samples in timecourse experiments. For each time point (0 hr to 180 dpi), five animals received a moderate (200 kd) contusion injury at T9, and spinal cord tissues were assessed by flow cytometry for the numbers of PMNs, ED1+ macrophages/microglia, and CD3+ T-cells. However, not all animal samples were recovered successfully for PMN (4-5), ED1 (3-5), and CD3 (4-5) flow cytometric analyses.
<img src="/files/ftp_upload/2698/2698fig4.jpg" alt="Figure 4"/> Figure 4. A timecourse of cellular inflammation in the spinal cord following a moderate (200 kd) contusion injury at T9. As assessed by flow cytometry, numbers of PMNs, ED1+ macrophages/microglia, and CD3+ T-cells peaked acutely (1,7, and 9 dpi, respectively) and persisted chronically in the injured spinal cord. n=3 to 5 per group, mean &plusmn; SEM.

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	<li>Beck, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cellular+inflammation+after+traumatic+spinal+cord+injury:+evidence+for+a+multiphasic+inflammatory+response+in+the+acute+to+chronic+environment.">Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment.</a> Brain. 133, 433-447 (2010).</li><li>Nguyen, H. X., Galvan, M. D., Anderson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+early+and+terminal+complement+proteins+associated+with+polymorphonuclear+leukocytes+in+vitro+and+in+vivo+after+spinal+cord+injury.">Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes in vitro and in vivo after spinal cord injury.</a> J Neuroinflammation. 5, 26-26 (2008).</li><li>Saville, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monoclonal+antibody+to+CD11d+reduces+the+inflammatory+infiltrate+into+the+injured+spinal+cord:+a+potential+neuroprotective+treatment.">A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment.</a> J Neuroimmunol. 156, 42-57 (2004).</li><li>Popovich, P. 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No conflicts of interest declared.

The use of flow cytometry to quantify immune cells in the CNS is complicated by lipid/myelin content and debris. In addition to interfering with antibody binding and specificity, myelin debris can have similar size and granulation properties to immune cells, decreasing measurement sensitivity and accuracy 8. The novel cell preparation method described here is effective in removing myelin debris and thereby improves the sensitivity of flow cytometry to detect changes in cellular inflammation in the injured spinal cord. For instance, the removal of myelin debris by this method enhanced detection of immune cells compared to enzymatic dissociation alone.Critically, novel usage of this method allowed the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for PMNs, macrophages/microglia, and T-cells up to 180 dpi, and identified a novel second phase of cellular inflammation. These important findings of a multiphasic component of neuroinflammation may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.
The OptiPrep gradient system has been used to separate debris from cells in blood 9, or purify neurons from the brain for cell culture purposes 10; however, we have modified and advanced this method system to separate myelin debris from spinal cord dissociated cells, and allowed rapid and more accurate quantification of cellular inflammation by flow cytometry. This method together with flow cytometry is likely to provide significant advancement in inflammation research after CNS injuries or pathological conditions, as most studies have only characterized cellular inflammation in the CNS using morphological and histological techniques that are not cell-type specific or cannot always provide true quantitative assessment. Furthermore, a few recent studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue 11,12 or cells separated by the Percoll gradient system 13; however, these studies have demonstrated either low cell yields or insensitive detection of immune cells.
In contrast, the OptiPrep gradient cell preparation method has provided for the first time, sensitive and reliable quantitative assessment by flow cytometry to changes of cellular inflammation in response to graded injury severity and over an extended timecourse up to 180 dpi. The sensitivity and reliability of flow cytometric analysis also depend on the specificity of antibodies used to detect specific immune cell types, as flow cytometry does not permit morphological criteria to further distinguish cell types. The specificity of antibodies for PMNs, macrophages/microglia or T-tells has been tested thoroughly in flow cytometry for peritoneal PMNs and alveolar macrophages in culture and cells in the injured spinal cord tissue 1,2. Together with the OptiPrep gradient system, these antibodies used for flow cytometry have contributed to the generation of a temporal and quantitative analysis of cellular inflammation that has provided new insight into the dynamics of the SCI microenvironment, and identified for the first time an extended multiphasic response of cellular inflammation. This multiphasic response of cells after SCI may be important to investigate the role of specific cell types present at specific times after injury or generate therapeutic interventions against specific cell types that can aggravate injuries. Furthermore, these findings may assist in the development of an appropriate rationale for the optimal drug- or cell-based interventions for SCI.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>OptiPrep</td>
 <td>Fisher Scientific</td>
 <td>NC9182490</td>
 </tr>
 <tr>
 	<td>HBSS</td>
 <td>Sigma</td>
 <td>H1387</td>
 </tr>

 <tr>
 	<td>Rabbit anti-rat PMN (FITC)</td>
 <td>Accurate Chemical &amp; Scientific</td>
 <td>AIFAD51140</td>
 </tr>

 <tr>
 	<td>Mouse anti-rat ED1 (Alexa Fluor 488)</td>
 <td>AbD Serotec</td>
 <td>MCA341A488</td>
 </tr>

 <tr>
 	<td>Mouse anti-rat CD3 (FITC)</td>
 <td>AbD Serotec</td>
 <td>MCA772F</td>
 </tr>

 <tr>
 	<td>Trypsin</td>
 <td>Sigma</td>
 <td>T9935</td>
 </tr>

 <tr>
 	<td>Collagenase</td>
 <td>Sigma</td>
 <td>C5138</td>
 </tr>

 <tr>
 	<td>DME</td>
 <td>Sigma</td>
 <td>D1152</td>
 </tr>

 <tr>
 	<td>MOUSE IgG1 (Alexa Fluor 488)</td>
 <td>AbD Serotec</td>
 <td>MCA1209A488</td>
 </tr>

 <tr>
 	<td>Fetal bovine serum</td>
 <td>Hyclone</td>
 <td>SH30070.03</td>
 </tr>

 <tr>
 	<td>Rabbit serum</td>
 <td>Jackson ImmunoResearch</td>
 <td>011-000-120011-000-120</td>
 </tr>

 <tr>
 	<td>Mouse serum</td>
 <td>Jackson ImmunoResearch</td>
 <td>015-000-120</td>
 </tr>

 <tr>
 	<td>Cell Strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 </tr>


 </tbody>

</table>

quantitative assessment of immune cells in the injured spinal cord tissue by flow cytometry:  a novel use for a cell purification method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Watch this Scientific Journal Video about Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method at JoVE.com

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

quantitative-assessment-immune-cells-injured-spinal-cord-tissue-flow

Research

JoVE Journal

Immunology and Infection

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, ...

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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 ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...