Questo lavoro è stato sostenuto dalla Società Nazionale Sclerosi Multipla (TA 3021-A1 / T per AEC) e l&#39;Università del Texas a San Antonio.

Preparazione dei reagenti Preparare magazzino isotonica Percoll (SIP), 4 ml al cervello, mescolando 9 parti di Percoll con una parte di HBSS 10X senza Ca + + e Mg + +. Preparare SIP al 70% in 1X HBSS senza Ca + + e Mg + +, 2 ml al cervello dispensato in un tubo da 15 ml in polipropilene. Nota: Si raccomanda di Percoll dovrebbe essere usato a temperatura ambiente, se usato a freddo, le cellule tendono a raggrupparsi e la separazione delle cellule è meno efficiente. Tessuto di raccolta e omogeneizzazione Anestetizzare topi e profumato attraverso il ventricolo sinistro con HBSS 1X ghiacciata. Sezionare il cervello e il midollo spinale e mantenere in RPMI senza rosso fenolo fino a quando tutti i topi vengono sacrificati. Tessuti posto in 7 o 15 ml dounce omogeneizzatore contenente 3 ml di RPMI, gentilmente fare una sospensione cellulare con una linea morbida pestello (Una dimensione) seguito da un Tigh-montaggio pestello (dimensione B) per dissociare ulteriormente i tessuti. Completa il volume della sospensione cellulare a 7 ml con RPMI. Gradiente impostato Aggiungere 3 ml di SIP alla sospensione cellulare per fare un SIP finale del 30% Lentamente lo strato di sospensione cellulare 10 ml sulla parte superiore della SIP 70%. Questa è la fase più critica della procedura, utilizzare una pipetta-aiuto al modo di gravità evitando la miscelazione del 70% e il 30% delle soluzioni. Una linea molto chiara piatto dovrebbe essere visibile al 70% -30% di giunzione. Centrifugare 30 min a 500G 18 ° C. Assicurarsi centrifuga si ferma con freno minimo o nessun modo che l&#39;interfase non è disturbato. Utilizzando una pipetta di trasferimento, rimuovere delicatamente lo strato di detriti dalla parte superiore del tubo e raccogliere 2,0-3,0 ml di 70% -30% interfase in una provetta pulita conica contenente 8 ml di 1X HBSS. Assicurarsi che il Percoll contenente l&#39;interfase viene diluita a circa tre volte, mescolare un paio di volte per inversione e centrifugre 7 min a 500G a 18 ° C. Risospendere il pellet in 1 ml di buffer di colorazione della cellula e trasferirlo in una provetta da 1,5 ml e lavare ancora una volta utilizzando un micro-centrifuga a 10.000 G 1 min a 4 ° C. Anticorpi colorazione Pellet Risospendere in 50 ml di purificato antimouse CD16/CD32 diluito 1:200 in tampone di colorazione delle cellule per bloccare i siti di legame Fc. Incubare sul ghiaccio per 10 minuti. Contare le cellule utilizzando un emocitometro. Aggiungere 50 ml di miscela di anticorpi cocktail e incubare in ghiaccio per 30 min. Ogni anticorpo deve essere il primo titolato a valutare la diluizione ottimale. Uso appropriato combinazioni fluorocromo scelti in base alla capacità di laser e impostazioni del filtro del citofluorimetro particolare. Lavare le cellule in tampone di colorazione della cellula, pellets risospendere in 100-200 ml di buffer di colorazione della cellula e di analizzare immediatamente nel citometro, o in alternativa le cellule possono essere risospese in 2% paraformaldeide (PFA), preparati in PBS e conservato durante la notte a 4 ° C . Rappresentante dei risultati: Dopo avere girato le pendenze, il 70% -30% interfase dovrebbe avere definito un alone bianco, e la quantificazione delle cellule recuperate da un mouse ingenuo deve cedere 3-5 x10 5 celle per il mouse (cervello più midollo spinale). Cervello infiammato potrebbe avere i numeri che variano a seconda delle cellule infiammatorie del sistema nervoso centrale insulto o modello di malattia (Figura 1). <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento di cellule mononucleate, da ingenuo e EAE-cervello a malattia di picco. Le cellule cerebrali sono stati separati sospensioni più discontinuo del 70% / pendenze Percoll 30%. Macchiato le cellule sono state incubate con Fc-block seguiti da anticorpi anti-CD45 per 30 minuti sul ghiaccio. Le cellule sono state lavate in tampone di colorazione della cellula, e fissato nel 2% PFA prima acquisizione in un LSR-II. CD45 intensità è misurata in asse Y, dove tre popolazioni distinte sono visualizzati. Un&#39;ulteriore caratterizzazione del fenotipo CD45 delle cellule immunitarie di alta infiltrazione è implementata utilizzando anticorpi aggiuntivi e successivamente analizzati mediante citometria di flusso.

<ol>
	<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>

Nessun conflitto di interessi dichiarati.

Le analisi di marcatori cellulari di superficie nei leucociti sistema nervoso centrale dai tessuti del mouse normale e infiammata è stata utilizzata per decenni 1-3. Protocolli di isolamento si basano nella separazione della microglia e leucociti da 4 densità centrifugazione. Qui si descrive un metodo veloce ed efficace di isolare leucociti sistema nervoso centrale mediante gradienti di Percoll discontinuo. Dopo l&#39;isolamento delle cellule, colorazione con anticorpi diversi come ad esempio-ma ...

Omogeneizzazione dei tessuti e gradienti <ul><li> Percoll (Amersham) </li><li> Sale 10X Hanks &#39;soluzione equilibrata, senza calcio e magnesio (Gibco) </li><li> RPMI Media 1640, senza rosso fenolo (Gibco) </li><li> 7-ml o 15 ml Dounce Tissue Smerigliatrici (VWR) </li></ul> Citometria a flusso <ul><li> Buffer di colorazione delle cellule (Biolegend) </li><li> Rat purificato CD16/CD32 anti-topo (mouse BD Fc Block), BD Pharmingen </li><li> Emocitometro (Fisher) </li><li> Anti-topo CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend </li><li> Soluzione salina tamponata con fosfato 10X, senza calcio e magnesio (Thermo Scientific) </li><li> Paraformaldeide (Sigma Aldrich) </li><li> Analisi di citometria a flusso eseguita con un II LSR (BD Biosciences) </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

Isolamento di cellule immunitarie che si infiltrano nel sistema nervoso centrale (SNC) durante l&#39;infezione, traumi, malattie autoimmuni o neurodegenerazione, è spesso necessaria per definire la loro fenotipo e funzioni effettrici. Approcci istochimiche sono strumentali per determinare la posizione delle celle di infiltrarsi e di analizzare il sistema nervoso centrale associata patologia. Tuttavia, in situ e tecniche di colorazione istochimica immunofluorescenza sono limitati dal numero di anticorpi che possono essere usati in una sola volta a caratterizzare sottotipi di cellule immunitarie in un particolare tessuto. Pertanto, gli approcci istologici in collaborazione con immuno-fenotipo mediante citometria di flusso sono fondamentali per caratterizzare completamente la composizione del locale infiltrazione sistema nervoso centrale. Questo protocollo si basa sulla separazione del sistema nervoso centrale sospensioni cellulari più discontinue gradienti di Percoll. L&#39;articolo attuale descrive un protocollo rapido per isolare in modo efficace le cellule mononucleate da tessuti cerebrali e del midollo spinale che può essere efficacemente utilizzato per l&#39;identificazione delle varie popolazioni di cellule immunitarie in un singolo campione mediante citometria di flusso.

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

L&#39;articolo attuale descrive un protocollo rapido per isolare in modo efficace le cellule mononucleate da tessuti cerebrali e del midollo spinale che può essere efficacemente utilizzato per le analisi di citometria a flusso.

Isolamento del cervello e cellule mononucleari del midollo spinale Uso dei gradienti Percoll

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

isolation-brain-spinal-cord-mononuclear-cells-using-percoll

Research

JoVE Journal

Immunology and Infection

40.5K Views.  University of Texas at San Antonio - UTSA. L'articolo attuale descrive un protocollo rapido per isolare in modo efficace le cellule mononucleate da tessuti cerebrali e del midollo spinale che può essere efficacemente utilizzato per le analisi di citometria a flusso.

Video: 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.

La valutazione quantitativa delle cellule immunitarie nel tessuto danneggiato del midollo spinale mediante citometria di flusso: un nuovo uso per un metodo di purificazione cellulare

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

Ricerca

Immunologia e infezioni

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.

Isolamento di Brain-infiltrazione Leucociti

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.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

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.

Isolamento e analisi citofluorimetrica di-Glioma infiltrazione sangue periferico mononucleari Cellule

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.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando 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 ...

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.

Isolamento delle cellule mononucleari del Tonsillar per studiare le risposte immunitarie innate Ex Vivo in un tessuto linfoide mucosale umano

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.

Isolamento, attivazione ed espansione delle cellule T basate sulla flottazione da campioni di cellule mononucleate del sangue periferico umano utilizzando microbolle

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

Raccolta e crioconservazione PBMC per analisi bioenergetiche mitocondriali

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.

Autore in primo piano: Conservazione dei parametri bioenergetici nelle cellule mononucleate del sangue periferico dopo la crioconservazione 

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.

Crioconservazione e valutazione bioenergetica delle cellule mononucleate del sangue periferico umano

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