<ol><li> Prepare canais microfluídicos com anti-CD4 + biotinlayted superfície. </li><li> Injectar 10 ml de amostra de sangue de 5 ml / min no canal microfluídicos com bomba de seringa. </li><li> Lavar o sangue de 20 ml / min para 20 min com PBS. </li><li> Injectar a solução diluída de lise FACS (diluído a 10% da solução estoque com PBS) em 5ul/min por 15 min. </li><li> Incubar por 15 min. </li><li> Injetar CD3 + solução de anticorpos primários (diluído a 10% da solução estoque com PBS) a 5 ml / min para 20 min. </li><li> Incubar por 2 horas. </li><li> Lave canal com PBS, 5 ml / min para 15 min. </li><li> Injectar 20% AF488 CD4 + de anticorpos, solução estoque 0,2% DAPI, e 10% CD3 + anticorpo secundário a 5 ml / min para 20 min, ao mesmo tempo. </li><li> Incubar por 20 min. </li><li> Lave o canal com PBS, 5 ml / min para 15 min. </li><li> Contagem de células ao microscópio fluorescente: <ol><li> White (óptica) - Preto e branco: célula e poeira </li><li> UV (DAPI) - Blue: única célula </li><li> Azul (CD4 +) - Verde: linfócitos T e monócitos </li><li> Verde (CD3 +) - Vermelho: linfócitos T só </li></ol></li></ol>

<table><tbody><tr><td>PDMS microchannel</td><td></td><td></td><td></td><td>Fabricated by SU-8 mold</td></tr><tr><td>Blood</td><td></td><td></td><td></td><td>Donated blood from normal human subject. Mass General Hospital, 10 X high density WBC or Brigham and Women&amp;rsquo;s Hospital.</td></tr><tr><td>PBS</td><td>Buffer</td><td>Invitrogen</td><td>14190</td><td>1x Dulbecco&amp;rsquo;s Phosphated Buffered Saline.</td></tr><tr><td>FACS lysing solution</td><td></td><td>BD Biosciences</td><td>349202</td><td>Diluted to 10% from stock solution with PBS.</td></tr><tr><td>Anti-CD4+ biotinlayted Ab</td><td></td><td>Beckman Coulter Inc.</td><td>IM0704</td><td>Surface prepared with Anti-CD4+ biotinlayted Ab, diluted to 10% from stock solution with PBS/BSA after mixing with 0.2mg stock and 1ml deionized water. </td></tr><tr><td>goat anti-hCD3delta</td><td>pAB</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-1128</td><td>Diluted to 10% from stock solution with PBS</td></tr><tr><td>rabbit anti-goat IgG PE</td><td></td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-3755</td><td>Secondary Ab fro anti-CD3 Ab, diluted to 10% from stock solution with PBS</td></tr><tr><td>anti-human CD4+</td><td></td><td>BD Biosciences</td><td>557695</td><td>Alexa Fluor 488 labeled, diluted to 20% from stock solution with PBS. </td></tr><tr><td>DAPI</td><td></td><td>Invitrogen</td><td>D1306</td><td>4&amp;rsquo;,6-dianidino-2-phenylindole, dihydrochloride solution, diluted to 0.2% from stock solution with PBS.</td></tr></tbody></table>

cd4+ t-lymphocyte capture using a disposable microfluidic chip for hiv

Watch this Scientific Journal Video about CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV at JoVE.com

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T-linfócitos Captura Usando um chip descartável microfluídicos para HIV

cd4-t-lymphocyte-capture-using-a-disposable-microfluidic-chip-for-hiv

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.6K visualizações.  Brigham and Women's Hospital. 

Vídeo: CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizando transferência de células-célula de HIV usando Clones fluorescente de HIV e Microscopia Confocal ao vivo

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Imunologia e Infecção

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

Biologia

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fabricação de chips microfluídicos e método para detectar a gripe

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Bioengenharia

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Um chip microfluídico para a Versátil Análise Química de células individuais

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plataforma microfluídico com multiplexada detecção electrónica do Ordenamento do Rastreamento de Partículas

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

De células-tronco Baseado Imunidade Engineered contra infecção por HIV no modelo humanizado mouse

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Medicina

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Modelos de Co-Cultura Microfluídica para Dissecar a Resposta Imune em Microambientes Tumorais in vitro

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Plataforma de chip microfluídico clínico para o isolamento de células tumorais circulantes versáteis

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Caracterização unicelular de influxo de cálcio e infecção pelo HIV-1 usando uma plataforma optofluidica multiparmétrica

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...