이 작품은 국립 다중 경화증 협회 (TA AEC에 3021 - A1 / T)와 산 안토니오 텍사스 대학에 의해 지원되었다.

시약의 준비 칼슘없이 10X HBSS의 한 부분과 percoll 9 부분을 혼합하여 주식 isotonic percoll (SIP), 뇌 당 4 ML을 준비 + +와 MG + +. 없이 1X HBSS 70 %로 SIP를 준비 칼슘 + +와 MG + +, 15 ML의 폴리 프로필렌 원뿔 관에 적절 뇌의 당 2 ML. 참고 :이 percoll은 실온에서 사용해야하는 것이 좋습니다, 감기를 사용한 경우, 세포 클럼프하는 경향과 세포 분리 덜 효율적이다. 조직 수집 및 균질화 얼음처럼 차가운 1X HBSS과 좌심실을 통해 마우스 및 perfuse를 마취. 뇌 및 척수를 해부하다 모든 생쥐가 희생되기 전까지는 페놀 레드없는 RPMI으로 유지합니다. 7 15 ML 다운스 호모 지 나이저는 RPMI 3 ML을 포함에서 개최 조직은 부드럽게 추가로 조직을 떼어 놓다하는 타이 맞는 유봉 (B 크기) 다음 느슨한 맞는 유봉 (크기)와 세포 현탁액합니다. RPMI 7 개의 ML에 셀 suspensions의 볼륨을 작성합니다. 그라데이션이 설정 최종 30 % SIP를 만들기 위해 세포 현탁액에 SIP 3 ML 추가 천천히 계층 70 % SIP 위에 10 ML 세포 현탁액. 이 절차의 가장 중요한 단계이며, 70 % 혼합하여 30 % 솔루션을 피하고 중력 모드에서 설정 피펫 에이드를 사용합니다. 명확 평평한 라인은 70 % -30 %의 교차점에 표시되어야합니다. 500G 18 ° C.에서 30 분 원심 분리기 원심 분리기는 계면이 방해되지 않도록 최소한의 또는 전혀 브레이크와 함께 중지됩니다 있는지 확인하십시오. 전송 pipet을 사용하면 부드럽게 튜브의 상단에서 파편의 레이어를 제거하고 1X HBSS 8 ML을 포함한 깨끗한 원뿔 관에 70 % -30 %의 계면의 2.0-3.0 ML를 수집합니다. 계면을 포함하고있는 percoll 세 배에 대한 희석되었는지 확인, 18 500G에서 역전과 centrifugre 7 분으로 몇 번 혼합 ° C. Resuspend 세포 얼룩 버퍼 1 ML의 펠렛 및 1.5 ML 튜브로 전송하고 4 만 G 1 분 ° C.에서 마이크로 원심 분리기를 사용하여 한 번 더 씻어 항체 염색법 antimouse CD16/CD32 투석을 50 μl의 Resuspend 알약은 FC 바인딩 사이트를 차단하는 세포 얼룩 버퍼에 1:200 희석. 10 분 얼음 위에 알을 품다. hemocytometer를 사용하여 셀을 계산합니다. 30 분 얼음 항체 칵테일 믹스와 부화 50 μl를 추가합니다. 각 항체 먼저 최적의 희석을 평가하기 위해 titrated해야합니다. 레이저와 특정 흐름 cytometer의 필터 설정의 기능에 따라 선택한 해당 fluorochrome 조합을 사용합니다. 세포 얼룩 버퍼의 100-200 μl의 세포 얼룩 버퍼, resuspend 알약의 세포를 세척하고 cytometer에 즉시 분석하거나, 또는 세포는 4 PBS의 준비 2 % paraformaldehyde (PFA)에 resuspended 이상 박 저장할 수 있습니다 ° C . 대표 결과 : 그라디언트를 회전 후, 70 % -30 %의 계면은 정의된 흰색 후광이 있어야하고, 순진한 마우스에서 발견한 세포의 quantitation 마우스 당 3-5 X10 5 세포 (뇌 척수 플러스) 양보해야합니다. 염증 두뇌 CNS의 모욕 또는 질병 모델 (그림 1)에 따라 염증 세포의 숫자까지 할 수도 있습니다. <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="그림 1" /> 그림 1. 피크 질병에 순진하고 EAE - 머리에서 mononuclear 세포의 분리. 뇌 세포의 suspensions는 불연속 70% / 30 % percoll의 그라디언트를 통해 분리되었다. 세포 얼룩이 얼음에 30 분 안티 CD45 항체 뒤에 FC - 블록 incubated되었습니다. 세포는 세포 얼룩 버퍼에 씻어서하고, LSR - II에 2% PFA 전에 인수에서 수정되었습니다. CD45 강도는 세 개의 서로 다른 인구가 시각의 Y - 축 단위로 측정됩니다. CD45 안녕 침투 세포의 면역 표현형의 추가 특성이 추가 항체를 사용하여 구현하고 이후 유동세포계측법 의해 분석됩니다.

<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>

관심 없음 충돌 선언하지 않습니다.

정상 및 염증 마우스 조직에서 CNS의 leukocytes의 세포 표면 마커의 분석은 1-3 몇 수십 년 동안 이용되었다. 격리 프로토콜은 밀도 원심 분리에 의해 4 microglia 및 leukocytes의 분리에 근거하고 있습니다. 여기 불연속 percoll의 그라디언트를 사용하여 CNS의 leukocytes를 격리의 신속하고 효과적인 방법을 설명합니다. 세포의 분리 후, 각종 항체와 얼룩으로 - 이에 - CD45 이에 국한되지 않음, CD4...

조직 균질화 및 그라디언트 <ul><li> Percoll (아머샴) </li><li> 칼슘과 마그네슘없이 10X 행크스 &#39;밸런스드 소금 솔루션 (Gibco) </li><li> 페놀 레드 않고 중간 RPMI 1640 (Gibco) </li><li> 7 - ML 15 - ML 다운스 조직 그라 인더스 (VWR) </li></ul> 유동세포계측법 <ul><li> 세포 스테 이닝 버퍼 (Biolegend) </li><li> 정화 랫 방지 마우스 CD16/CD32 (마우스 BD FC 블록), BD Pharmingen </li><li> Hemacytometer (피셔) </li><li> 안티 마우스 CD45 (복제 30 - F11), CD4 (클론 RM4 - 5), CD19 (클론 6D5), BioLegend </li><li> 10X 인산은 칼슘과 마그네슘없이 살린 버퍼 (써모 과학) </li><li> Paraformaldehyde (시그마 알드리치) </li><li> 유동세포계측법 분석 LSR II (BD Biosciences)와 수행 </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

감염, 외상, autoimmunity 또는 neurodegeneration 동안 중추 신경계 (CNS)를 침투 면역 세포의 분리는 종종 자신의 표현형 및 이펙터 기능을 정의해야합니다. Histochemical 접근 침투 세포의 위치를​​ 파악하고 병리 관련 CNS를 분석하는 수단입니다. 그러나, 원위치 histochemistry과 immunofluorescent 얼룩 기술은 특정 조직에 면역 세포 subtypes을 특성화하기 위해 한 번에 사용할 수있는 항체의 숫자에 의해 제한됩니다. 따라서, 유동세포계측법에 의해 면역 - phenotyping와 함께 histological 접근 방법이 완전히 지역 CNS의 침투의 조성을 특성화하기 위해 중요하다. 이 프로토콜은 discontinous percoll 그라디언트를 통해 CNS 세포 suspensions의 분리에 따라 달라집니다. 현재 문서는 효율적으로 효과적으로 유동세포계측법하여 하나의 샘플에서 다양한 면역 세포 집단의 식별을 위해 활용할 수있는 두뇌 및 척수 조직에서 mononuclear 세포를 분리하기 위해 빠른 프로토콜을 설명합니다.

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

현재 문서는 효율적으로 효과적으로 흐름 cytometric 분석을 위해 활용할 수 뇌 및 척수 조직에서 mononuclear 세포를 분리하기 위해 빠른 프로토콜을 설명합니다.

Percoll 그라디언트를 사용하여 두뇌와 척수 Mononuclear 세포의 분리

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 조회수.  University of Texas at San Antonio - UTSA. 현재 문서는 효율적으로 효과적으로 흐름 cytometric 분석을 위해 활용할 수 뇌 및 척수 조직에서 mononuclear 세포를 분리하기 위해 빠른 프로토콜을 설명합니다.

비디오: 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.

유동세포계측법에 의해 피해를 입은 척수 조직에서 면역 세포의 양적 평가 : 세포 정제 방법에 대한 소설을 사용

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

연구

면역학 및 감염병학

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.

브레인 - 침투 Leukocytes의 분리

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.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

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.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

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.

콜라게나아제 E를 사용하여 뮤린 결장에서 라미나 프로프리아 단핵 세포의 분리

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.

편도선 단핵 세포의 격리는 인간 점막 림프구 조직에서 Ex Vivo 타고난 면역 반응을 연구합니다

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.

부유 기반 T 세포 분리, 활성화 및 마이크로버블을 사용한 인간 말초 혈액 단핵 세포 샘플로부터의 확장

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

미토콘드리아 생물 에너지 분석을 위한 PBMC 수집 및 냉동 보관

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.

저자 스포트라이트: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (냉동 보존 후 말초 혈액 단핵 세포의 생체 에너지 매개변수 보존) 

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.

인간 말초 혈액 단핵 세포의 냉동 보존 및 생체 에너지 평가

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