Este trabajo fue financiado por los Institutos Nacionales de Salud de subvención: PO1 HL55798 (a KL) y la American Heart Association de Desarrollo Científico subvención 0525532U (a EG).

<ol>
	<li>Lusis, 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=Atherosclerosis.">Atherosclerosis.</a> Nature. 407, 233-2341 (2000).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+and+inflammatory+mechanisms+of+atherosclerosis.">Immune and inflammatory mechanisms of atherosclerosis.</a> Annu Rev Immunol. 27, 165-197 (2009).</li><li>Hansson, G. K., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+in+atherosclerosis:+a+double-edged+sword.">The immune response in atherosclerosis: a double-edged sword.</a> Nat Rev Immunol. 6, 508-519 (2006).</li><li>Galkina, E., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+influx+in+atherosclerosis.">Leukocyte influx in atherosclerosis.</a> Curr Drug Targets. 812, 1239-1248 (2007).</li><li>Bonanno, E., Mauriello, A., Partenzi, A., Anemona, L., Spagnoli, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+atherosclerotic+plaque+cells+from+human+carotids:+a+validation+study.">Flow cytometry analysis of atherosclerotic plaque cells from human carotids: a validation study.</a> Cytometry. 39, 158-165 (2000).</li><li>Liu-Wu, Y., Svenningsson, A., Stemme, S., Holm, J., Wiklund, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+macrophage-derived+foam+cells+from+human+atherosclerotic+lesions+by+using+a+"mock"+FL3+channel+in+flow+cytometry.">Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.</a> Cytometry. 29, 155-164 (1997).</li><li>Galkina, E., Kadl, A., Sanders, J., Varughese, D., Sarembock, I. J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+recruitment+into+the+aortic+wall+before+and+during+development+of+atherosclerosis+is+partially+L-selectin+dependent.">Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.</a> J Exp Med. 203, 1273-1282 (2006).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. 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><li>Smith, E., Prasad, K. M., Butcher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+interleukin-17A+results+in+reduced+atherosclerosis+in+apolipoprotein+E-deficient+mice.">Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice.</a> Circulation. 121, 1746-1755 (2010).</li><li>Eid, R. E., Rao, D. A., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17+and+interferon-gamma+are+produced+concomitantly+by+human+coronary+artery-infiltrating+T+cells+and+act+synergistically+on+vascular+smooth+muscle+cells.">Interleukin-17 and interferon-gamma are produced concomitantly by human coronary artery-infiltrating T cells and act synergistically on vascular smooth muscle cells.</a> Circulation. 119, 1424-1432 (2009).</li></ol>

A continuación, presentamos un método de la citometría de flujo basado en la investigación de la composición de las células inmunes de aortas murino. La principal ventaja de este método es la capacidad de analizar la aorta células del sistema inmune en un nivel de células individuales y para caracterizar el estado de activación de los leucocitos aórtica. Este método no se limita a aortas murino y nosotros (datos no publicados) y otras 10 personas utiliza este enfoque para analizar las muestras hum...

<table border="1"><tbody><tr><td> Material </td><td> Número de catálogo </td><td> Empresa </td></tr><tr><td> Colagenasa XI </td><td> C7657 </td><td> Sigma-Aldrich </td></tr><tr><td> Hialuronidasa </td><td> H3506 </td><td> Sigma-Aldrich </td></tr><tr><td> DNasa I, tipo 2 </td><td> D4527 </td><td> Sigma-Aldrich </td></tr><tr><td> Colagenasa I </td><td> C0130 </td><td> Sigma-Aldrich </td></tr><tr><td> Colagenasa II </td><td> LS004174 </td><td> Worthington Biochemical Corp. </td></tr><tr><td> Elastasa </td><td> LS002292 </td><td> Worthington Biochemical Corp. </td></tr></tbody></table>

flow cytometry analysis of immune cells within murine aortas

La aterosclerosis es un proceso inflamatorio crónico de los barcos de tamaño medio y grande que se caracteriza por la formación de placas consiste en células espumosas, células del sistema inmune, las células vasculares endoteliales y de músculo liso, plaquetas, la matriz extracelular, y un núcleo rico en lípidos, con extensa necrosis y fibrosis de los tejidos circundantes. 1 Los brazos innata y adaptativa de la respuesta inmune están implicados en la iniciación, desarrollo y la persistencia de la aterosclerosis. 2, 3 hay un importante cuerpo de evidencia de que los diferentes subconjuntos de las células inmunes, como los macrófagos, dendríticas las células, los linfocitos T y B, están presentes en las aortas de los ratones sanos y con tendencia aterosclerosis-4. Además, las células inmunes se encuentran en la adventicia aórtica rodea lo que sugiere un papel importante de este tejido en la aterogénesis. 2 Desde hace algún tiempo, la detección cuantitativa de los diferentes tipos de células del sistema inmune, su estado de activación, y la composición celular dentro de la pared de la aorta se vio limitada por RT-PCR y los métodos de inmunohistoquímica para el estudio de la aterosclerosis. Pocos intentos se hicieron para realizar citometría de flujo utilizando aortas humanos, y una serie de problemas, tales como una alta autofluorescencia, se han reportado 5,6. Las placas ateroscleróticas humanas fueron digeridos con colagenasa 1, y se recogieron las células libres y se tiñeron de CD14 + / + CD11c para destacar derivada de macrófagos en células espumosas. En este estudio, un &quot;simulacro&quot; de canal se utiliza para evitar falsos positivos tinción. 6 material necrótico acumulado durante el proceso de digestión dar lugar a una gran cantidad de desechos que genera una alta autofluorescencia en las muestras de la aorta. Para resolver este problema, un panel de controles positivos y negativos se ha propuesto, pero sólo la doble tinción se podría aplicar en estas muestras. Hemos desarrollado un nuevo flujo de citometría basado en el método 7 para analizar la composición de las células inmunitarias y caracterizar la activación, proliferación, diferenciación de las células inmunes en aorta sana y propenso aterosclerosis. Este método permite la investigación de la composición de las células inmunes de la pared aórtica y abre la posibilidad de utilizar una amplia gama de métodos inmunológicos para la investigación de los aspectos inmunológicos de la enfermedad.

Watch this Scientific Journal Video about Flow Cytometry Analysis of Immune Cells Within Murine Aortas at JoVE.com

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Este trabajo presenta una citometría de flujo basado en el método para investigar la composición inmunológica de aortas. El documento también muestra una técnica adicional que permite examinar los alrededores y adventicia pared de los vasos por separado. Este método abre la posibilidad de realizar análisis fenotípico de los leucocitos aórtica y aplicar varios ensayos inmunológicos para los estudios de la aterosclerosis.

Análisis por citometría de flujo de células inmunes dentro de aortas murino

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

flow-cytometry-analysis-of-immune-cells-within-murine-aortas

Research

JoVE Journal

Immunology and Infection

34.3K vistas.  Eastern Virginia Medical School. Este trabajo presenta una citometría de flujo basado en el método para investigar la composición inmunológica de aortas. El documento también muestra una técnica adicional que permite examinar los alrededores y adventicia pared de los vasos por separado. Este método abre la posibilidad de realizar análisis fenotípico de los leucocitos aórtica y aplicar varios ensayos inmunológicos para los estudios de la aterosclerosis.

Video: Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

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

Investigación

Inmunología e Infección

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.

Humano In Vitro Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

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.

Análisis cualitativo y cuantitativo de la sinapsis inmune en el sistema humano utilizando imágenes de citometría de flujo

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.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

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

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discriminación de siete subconjuntos de células inmunes por dos fluorocromo citometría de flujo

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparación de la médula ósea entera para el análisis de citometría de masa de células de linaje neutrófilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Aislamiento de células CD8 + T residentes en el riñón delratón para el análisis de citometría de flujo

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Análisis de la restricción SAMHD1 por citometría de flujo en células mieloides humanas U937

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...