Diese Arbeit wurde von der National Multiple Sclerosis Society (TA 3021-A1 / T, um AEC) und der University of Texas at San Antonio unterstützt.

Vorbereitung der Reagenzien Bereiten Lager isotonischen Percoll (SIP), 4 ml pro Gehirn, indem man 9 Teile Percoll mit einem Teil 10X HBSS ohne Ca + + und Mg + +. Bereiten Sie SIP bei 70% in 1X HBSS ohne Ca + + und Mg + +, 2 ml pro Kopf in einen 15 ml-Polypropylen-konischen Rohr verzichtet. Hinweis: Es wird empfohlen, Percoll bei Raumtemperatur verwendet werden sollte, falls verwendet Kälte, neigen Zellen zu verklumpen und Zellseparation ist weniger effizient. Tissue-Sammlung und Homogenisierung Anesthetize Mäusen und perfuse durch den linken Ventrikel mit eiskaltem 1X HBSS. Dissect Gehirn und Rückenmark und zu pflegen in RPMI ohne Phenolrot, bis alle Mäuse getötet werden. Legen Gewebe in 7 oder 15 ml Dounce Homogenisator mit 3 ml RPMI sanft eine Zellsuspension mit einer locker sitzenden Pistill (A-Größe) durch eine Oberschenkel-Montage Stößel (B Größe) zur weiteren distanzieren Gewebe gefolgt. Füllen Sie das Volumen des Zell-Suspensionen zu 7 ml mit RPMI. Gradient einrichten 3 ml SIP zu der Zellsuspension zu einer endgültigen 30% SIP machen Langsam Schicht der 10 mL Zellsuspension auf dem 70% SIP. Dies ist der wichtigste Schritt des Verfahrens, Verwendung einer Pipette-Hilfe in der Schwere-Modus zu vermeiden Mischen von 70% und 30%-Lösungen gesetzt. Eine sehr klare flache Linie sichtbar sein soll an die 70% -30%-Übergang. Zentrifuge 30 min bei 500G 18 ° C. Stellen Sie sicher, Zentrifuge wird mit minimalen oder keinen Bremse zu stoppen, so dass die Interphase nicht gestört wird. Mit einem Transfer-Pipette vorsichtig ab, die Schicht der Ablagerungen aus dem oberen Ende des Röhrchens sammeln und 2,0-3,0 ml der 70% -30% der Interphase in ein sauberes konisches Rohr mit 8 ml 1X HBSS. Stellen Sie sicher, dass die Percoll mit der Interphase etwa das Dreifache verdünnt wird, mischen ein paar Mal durch Umdrehen und centrifugre 7 min bei 500G bei 18 ° C. Pellet in 1 ml der Zell-Färbepuffer und überträgt es auf eine 1,5 ml-Tube und waschen ein weiteres Mal mit einem Mikro-Zentrifuge bei 10.000 G 1 min bei 4 ° C. Antikörper-Färbung Resuspendieren Pellets in 50 ul des gereinigten anti-Maus CD16/CD32 verdünnt 1:200 in Zelle Färbepuffer an den Fc-Bindungsstellen zu blockieren. Inkubieren auf Eis für 10 min. Zählen von Zellen mit einer Zählkammer. Add 50 ul Antikörper-Cocktail-Mix und Inkubation auf Eis für 30 min. Jeder Antikörper zunächst titriert werden, um eine optimale Verdünnung zu beurteilen. Verwenden Sie geeignete Fluorochrom Kombinationen ausgewählt nach den Möglichkeiten der Laser-und Filter-Einstellungen Ihres jeweiligen Durchflusszytometer. Wash-Zellen in Zellen Färbepuffer, resuspendieren Pellets in 100-200 ul der Zelle Färbepuffer und analysieren sofort im Durchflusszytometer oder alternativ Zellen können in 2% Paraformaldehyd (PFA) in PBS resuspendiert vorbereitet und gespeichert werden über Nacht bei 4 ° C . Repräsentative Ergebnisse: Nach dem Spinnen der Gradienten, sollte die 70% -30% der Interphase haben einen definierten weißen Heiligenschein, und die Quantifizierung der Zellen aus einer naiven Maus erholt sollte 3-5 x10 5 Zellen pro Maus (Gehirn und Rückenmark) zu ergeben. Entzündete Gehirn haben könnte reichen Zahl der Entzündungszellen in Abhängigkeit von der ZNS-Beleidigung oder Krankheit Modell (Abbildung 1). <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Isolierung von mononukleären Zellen aus naiven und EAE-Hirn-zu Spitzenzeiten Krankheit. Gehirnzellen Suspensionen wurden über diskontinuierliche 70% / 30% Percoll Gradienten getrennt. Zellen gefärbt wurden mit Fc-Block inkubiert, gefolgt von anti-CD45-Antikörper für 30 min auf Eis. Die Zellen wurden in Zellfärbung Puffer gespült und fest in 2% PFA vor Übernahme in ein LSR-II. CD45 Intensität ist in der Y-Achse, wo drei unterschiedliche Populationen visualisiert werden gemessen. Die weitere Charakterisierung der Immunabwehr Phänotyp CD45 hallo infiltrierenden Zellen erfolgt über zusätzliche Antikörper und anschließend mittels Durchflusszytometrie analysiert.

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	<li>Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154, 4309-4321 (1995).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Juedes, A. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+infiltrating+central+nervous+system+APCs+regulate+the+emergence+and+resolution+of+experimental+autoimmune+encephalomyelitis.">Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis.</a> J. Immunol. 166, 5168-5175 (2001).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tickets+to+the+brain:+Role+of+CCR2+and+CX(3)CR1+in+myeloid+cell+entry+in+the+CNS.">Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS.</a> J Neuroimmunol. , (2010).</li><li>Mildner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat. Neurosci. 10, 1544-1553 (2007).</li><li>Djukic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+monocytes+engraft+in+the+brain,+differentiate+into+microglia+and+contribute+to+the+pathology+following+meningitis+in+mice.">Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice.</a> Brain. 129, 2394-2403 (2006).</li></ol>

Analysen von Zelloberflächenmarker in CNS Leukozyten aus normalen und entzündeten Mausgeweben seit mehreren Jahrzehnten 1-3 verwendet worden. Isolation Protokolle sind in der Trennung von Mikroglia und Leukozyten durch Dichtezentrifugation 4. Hier beschreiben wir eine schnelle und effektive Methode zur Isolierung von CNS Leukozyten mit diskontinuierlichen Percoll-Gradienten. Nach der Isolierung von Zellen, Färbung mit verschiedenen Antikörpern wie, aber nicht beschränkt auf-CD45 ermöglicht CD...

Tissue Homogenisierung und Farbverläufe <ul><li> Percoll (Amersham) </li><li> 10x Hanks Balanced Salt Solution, ohne Calcium und Magnesium (Gibco) </li><li> RPMI Medium 1640, ohne Phenolrot (Gibco) </li><li> 7-ml-oder 15-ml Dounce Tissue Grinder (VWR) </li></ul> Durchflusszytometrie <ul><li> Zellfärbung Buffer (Biolegend) </li><li> Gereinigtem Ratten Anti-Mouse CD16/CD32 (Maus BD Fc Block), BD Pharmingen </li><li> Hämazytometer (Fisher) </li><li> Anti-Maus-CD45 (Klon 30-F11), CD4 (Klon RM4-5), CD19 (Klon 6D5), BioLegend </li><li> 10X Phosphate Buffered Saline, ohne Calcium und Magnesium (Thermo Scientific) </li><li> Paraformaldehyd (Sigma Aldrich) </li><li> Durchflusszytometrie-Analyse mit einer LSR II (BD Biosciences) durchgeführt </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

Isolierung von Immunzellen, die das zentrale Nervensystem (ZNS) während der Infektion, Trauma, Autoimmunität oder Neurodegeneration zu infiltrieren, ist oft erforderlich, um ihren Phänotyp und Effektor-Funktionen zu definieren. Histochemische Ansätze sind instrumental, um die Lage der infiltrierenden Zellen zu bestimmen und die damit verbundenen ZNS-Pathologie zu analysieren. Allerdings sind in-situ Histochemie und Immunfluoreszenzfärbung Techniken durch die Anzahl der Antikörper, die zu einem einzigen Zeitpunkt genutzt werden, um Immunzellen Subtypen in einem bestimmten Gewebe zu charakterisieren können, beschränkt. Daher sind histologische Ansätze in Verbindung mit Immun-Phänotypisierung mittels Durchflusszytometrie kritischen vollständig zu charakterisieren die Zusammensetzung der lokalen ZNS Infiltration. Dieses Protokoll basiert auf der Trennung von ZNS-Zellsuspensionen über diskontinuierliche Percoll Gradienten basiert. Der aktuelle Artikel beschreibt eine schnelle Protokoll zur effizienten Isolierung mononukleärer Zellen aus Gehirn und Rückenmark Gewebe, die effektiv für die Identifizierung von verschiedenen Immunzellen Populationen in einer einzigen Probe mittels Durchflusszytometrie verwendet werden kann.

Watch this Scientific Journal Video about Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients at JoVE.com

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Der aktuelle Artikel beschreibt eine schnelle Protokoll zur effizienten Isolierung mononukleärer Zellen aus Gehirn und Rückenmark Gewebe, die effektiv für den durchflusszytometrischen Analysen genutzt werden können.

Isolierung von Brain and Spinal Cord mononukleären Zellen mit Percoll Gradienten

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

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Research

JoVE Journal

Immunology and Infection

40.5K Aufrufe.  University of Texas at San Antonio - UTSA. Der aktuelle Artikel beschreibt eine schnelle Protokoll zur effizienten Isolierung mononukleärer Zellen aus Gehirn und Rückenmark Gewebe, die effektiv für den durchflusszytometrischen Analysen genutzt werden können.

Serie: Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

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

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

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

Quantitative Beurteilung der Immunzellen in das verletzte Rückenmark Tissue mittels Durchflusszytometrie: eine neue Verwendung für eine Zelle Reinigungsverfahren

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

Forschung

Immunologie und Infektion

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

Isolierung von Brain-infiltrierenden Leukozyten

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

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.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche 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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolierung und Analyse über Durchflusszytometrie von Gliom-Infiltration peripheren mononukleären Blutzellen

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolierung von Lamina Propria Mononukleären Zellen aus murine Colon mit Kollagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolierung von Tonsillar Mononukleären Zellen zur Untersuchung von Ex-Vivo-angeborenen Immunantworten in einem menschlichen Mukosal-Lymphgewebe

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Flotationsbasierte T-Zell-Isolierung, -Aktivierung und -Expansion aus mononukleären Blutproben des menschlichen peripheren Blutes unter Verwendung von Mikrobläschen

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

PBMC-Sammlung und Kryolagerung für die mitochondriale Bioenergetikanalyse

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Autor im Rampenlicht: Konservierung bioenergetischer Parameter in mononukleären Zellen des peripheren Blutes nach Kryokonservierung 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Kryokonservierung und bioenergetische Bewertung von mononukleären Zellen des menschlichen peripheren Blutes

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...