Name: isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue
Uploaded: 2018-07-17T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

The authors wish to acknowledge Becky Marquez, the clinical coordinator at the Sentara Bariatric Center, for her assistance with the process of patient screening and consenting. This research was supported by R15HL114062 to Anca D. Dobrian.

The Institutional Review Board Committee at Eastern Virginia Medical School approved the research and collection of human adipose tissue samples used in the study. Informed written consent was collected from the patients.
1. Preparation of Buffers, Media, and Instruments
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	<li>Prepare a Krebs Ringer Bicarbonate-Buffered Solution (KRBBS): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1 mM adenosine, 0.01% antib...

<ol>
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The focus of this paper is to provide a methodology for the isolation, expansion and adipogenic induction of CD34+CD31+ endothelial cells from visceral and subcutaneous depots of human adipose tissue.
Methodologies have been reported for the isolation of endothelial cells from various vascular beds of rodents or humans that involve primarily techniques using CD31 antibodies either fluorescently labeled or coupled to magnetic beads18,19...

Recent evidence shows that both in mice and in humans, a subset of cells residing in the adipose tissue vasculature can be differentiated into either white or beige/brown adipocytes1,2,3. The phenotype of such cells is a subject of controversy, with evidence supporting endothelial cells, smooth muscle/pericyte, or a spectrum of intermediate phenotypes4,5,6,7. The scope of developing this methodology was to test the adipogenic potential of CD34+CD31+ endothelial cells isolated from different fat depots from obese humans. Other studies in the literature are focusing on the adipogenic potential of the total stromal vascular fraction or of the known adipocyte progenitors2,8,9. Since currently existing technologies can target specifically adipose tissue endothelial cells for drug delivery10, understanding the potential of such cells to undergo adipogenic induction towards white or beige adipocytes is important for future targeted therapies.
Different groups reported the combination of CD31 and CD34 markers as surrogates to isolate endothelial cells from human adipose tissue11,12,13. Typically, the isolation is performed using two sequential steps and a positive selection using magnetic beads. In this report, immunoseparation using CD34+ magnetic beads combined with CD31 plastic beads was utilized. We found this technique superior to the sequential magnetic immunoseparation with respect to the preservation of typical cobblestone endothelial morphology. Also, we were able to generate enough cells required for the expansion and adipogenic induction starting from as little as 1&#8211;2 g of fat. A small sample biopsy of subcutaneous fat is enough to produce the required quantity of cells for downstream applications. This aspect is potentially important, particularly if this method will be utilized for screening for a responsiveness to adipogenic induction in human subjects.
Unlike other systems reported in the literature, this method utilizes only two ingredients for the adipogenic induction of the CD34+CD31+ cells: a PPAR&#947; agonist&#8212;rosiglitazone&#8212;and human insulin. Importantly, the amount of insulin used falls within the normal/high range of circulating post-absorptive insulin in humans14. The degree of responsiveness to insulin of the cells in vitro, measured by Akt phosphorylation, does not correlate with their ability to respond to the induction cocktail. Interestingly, using this induction cocktail and experimental conditions, a mix of white and beige/brown cells were obtained as determined by the size and numbers of intracellular lipid droplets and the expression of molecular markers. This straightforward and cost-effective induction protocol along with the quantitative evaluation of the phenotype of the responder cells (white vs. beige) allows for a screening of agents that can potentially alter the balance of differentiated beige:white adipocytes.
This method also provides a translational approach for understanding the underlying mechanisms of adipogenesis of vascular endothelial progenitors in human adipose tissue. Using this specific isolation/differentiation technique, investigators can interrogate various pathways responsible for adipogenesis in a subset of vascular endothelial cells from various fat depots in lean and obese humans.

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

Our protocol aims to provide an in vitro approach to determine the adipogenic potential of CD34+CD31+ vascular cells from different depots of human adipose tissue. A simplified flowchart diagram is shown in Figure 1A. The first step using a positive selection of CD34 expressing cells results in &#62; 95% CD34+ cells in the population of the freshly isolated cells (Figure 1A). Importantly, this marker is lost after the ce...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

This method can help answer key questions in the obesity field, such as identifying the key factors and mechanisms that prevent a robust adipogenic response during adipose tissue remodeling. The main advantage of this technique is that it allows isolation of CD34+CD31+endothelial cells from human adipose tissue starting with small amounts of tissue. We first had the idea for this method when we wanted to identify cells in human adipose vasculature that respond to adipogenic stimulation.Visual demonstration of this method is critical, as the cell isolation steps are difficult to practice due to the oftentimes limited access to fresh human adipose tissue. Collect human omental and subcutaneous adipose tissue from human subjects undergoing bariatric surgery. Immediately after tissue extraction, keep the human omental and subcutaneous adipose tissue in separate vials containing Hank's buffered salt solution with 50 micrograms per milliliter of penicillin streptomycin at room temperature.Once the sample arrives at the lab after the tissue extraction, carefully clean and remove fibrotic and cauterized sections of the tissue by using 70%ethanol-swabbed scissors and tweezers. Weigh the adipose tissue and partition it into five gram aliquots for the collagenase digestion. Use two pairs of scissors to finely mince five grams of the adipose tissue in five milliliters of a collagenase solution in a scintillation vial at room temperature.Add an additional 10 milliliters of collagenase solution to the minced tissue for 15 milliliters total. Digest the samples for one hour at 37 degrees Celsius in a water bath with continuous shaking. Following digestion, pour the samples into the 20 milliliter blunt end syringe.Filter the sample through 250 micron nylon mesh into the 50 milliliter conical tube to separate adipocytes and stromal vascular cells from undigested tissue. Wash out the scintillation vial with 10 milliliters of KRBBS, and pour the wash through the filter to collect residual cells before incubating the filtered samples for five minutes at room temperature. Then, utilize a 20 milliliter syringe and a 20 gauge by six inch pipetting needle to remove stromal vascular fraction layer and transfer it to a clean 50 milliliter conical tube.Add 10 milliliters of KRBBS and allow the samples to sit on the bench for five minutes at room temperature. Keep the stromal vascular fractions from both depots on ice for further processing. Spin the stromal vascular fractions at 500 times G for five minutes at 4 degrees Celsius.Remove the samples from the centrifuge and carefully pour off the supernatant, keeping the pellet containing the stromal vascular fraction cells. Gently re-suspend the cell pellets in five milliliters of PBS. After spinning the samples again, remove the supernatant, re-suspend the cells in one milliliter of PBS, and count the cells.Pellet the samples for a third time, and re-suspend the pellet in 100 microliters of CD34 isolation buffer. Add 10 microliters of CD34 cocktail and incubate the sample for 15 minutes at room temperature. Then add five microliters of magnetic beads and incubate the sample for 10 minutes at room temperature.Next, add 2.5 milliliters of CD34 isolation buffer to each sample, and place the samples into the magnet for five minutes at room temperature. Invert the magnet for five seconds to pour off the isolation buffer. After repeating this process four times, remove the tube from the magnet and add two milliliters of CD34 isolation buffer.Following another centrifugation, re-suspend the CD34+cell pellets in one milliliter of CD34 isolation buffer and count the cells. Pellet the cells again, and re-suspend the pellet in 250 microliters of Buffer B and 250 microliters of wash buffer. Next, thoroughly re-suspend the CD31 beads by vortexing the tube, then add 20 microliters of CD31 beads to the re-suspended cell pellet and incubate the sample with rocking for 30 minutes at room temperature.Prior to the cell separation, add one milliliter of wash buffer to equilabrate the strainer. Then, apply the prepared mixture of re-suspended cells in CD31 beads to the strainer. Wash the strainer with five milliliters of wash buffer in a circular motion for a total volume of 20 milliliters.Attach the connector to a sterile 50 milliliter conical tube and close the leur lock. After attaching the strainer to the connector, add one milliliter of wash buffer along the wall of the strainer, then add one milliliter of activated Buffer D along the wall of the strainer. Swirl the sample gently, and incubate it for 10 minutes at room temperature.Next, add one milliliter of wash buffer and separate the cells from the beads by pipetting up and down 10 times. Open the leur lock and allow the detached cells to flow into the 50 milliliter conical tube. Wash the strainer 10 times with one milliliter of wash buffer each time.Discard the connector and strainer, and centrifuge the samples at 300 times G for 10 minutes at 4 degrees Celsius. Finally, re-suspend the cell pellet in two milliliters of complete EGM2 media for culture. Grow the cells in six-well tissue culture treated plates with complete EGM2 media in a 37 degrees Celsius, 5%carbon dioxide incubator.Replace the media every three to four days until the cells reach 80 to 90%confluence. Then, seed approximately 100 to 200 thousand cells into six-well plates, ensuring duplicate wells for each depot, and culture them in complete EGM2 media for two days. After two days, aspirate off any growth media.Replace the media with two milliliters of DMEM/F-12 media with 5%FBS and 50 micrograms per milliliter of penicillin streptomycin for the control cells. For the treatment cells, replace the media with two milliliters of adipogenesis media. Incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 13 days replacing the respective media every three to four days.Representative results showing the typical endothelial cobblestone-like morphology of the CD34+CD31+cells after two passages in culture is shown. Also demonstrated is the ability of cells to form tubules when cultured in Matrigel as expected. Uniform uptake of fluorescently labeled acetylated LDL shows the presence of a homogenous cell population with endothelial identity.Shown here is Oil Red O staining of neutral lipid droplets in CD34+CD31+cells following 13 days of adipogenic induction. Representative responses of cells from omental and subcutaneous depots of three different human subjects are shown to illustrate the large variability in response to adipogenic induction between fat depots of the same donor and between donors. This image displays fluorescent intracellular lipid staining in CD34+C31+cells after adipogenic induction for 13 days.The image shows cells with unilocular lipids, multilocular lipids, and no lipid accumulation. Real time PCR shows the expression of the pan adipocyte marker adiponectin and the brown beige markers CIDEA and UCP-1. These results confirm that cells following adipogenic induction express molecular markers consistent with a mixed white beige adipocyte phenotype.Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember to use fresh human adipose tissue that was collected and not stored longer than two hours. Following this procedure, other methods like flow cytometry, immunohistochemistry, and RT-PCR can be performed to identify specific subsets of responding cells, and expression of mature adipocyte markers that can be utilized to identify a beige brown adipocyte phenotype.After it's development, this technique paved the way for researchers in the filed of obesity to explore roles of adipose tissue, vascular cells, and adipogenesis in humans. After watching this video, you should have a good understanding of how to isolate, culture, and expand CD34+31+cells. Don't forget that working with human tissues can be extremely hazardous, and precautions, such as bio-safety regulations, should always be taken while performing this procedure.The implications of this technique extend toward therapy of obesity associated comorbidities, such as insulin resistance, because it identifies adipogenic response of cells to a potential therapeutic intervention.

isolation-expansion-adipogenic-induction-cd34-cd31-endothelial-cells

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.