이 연구는 연구-CUHK (2016.035), 및 홍콩 학자에 대 한 연구 보조금 위원회의 홍콩 (GRF 468513, CUHK3/CRF/12R) 및 혁신 및 기술 기금의 홍콩 (ITS/227/15, ITS InP/164/16, ITS-InP/242/16), 직접 보조금에 의해 지원 되었다 프로그램입니다.
H. Y.L. 설계 및 모든 실험을 감독 및 원고 준비에 기여. 오후-K.T. 실험 수행, 데이터 분석 및 원고 준비에 기여. 시공-T. 토니, 재-F.C., M.W.-대표-C., 반장 님, 질문, 및 G. Y.L. 동물 샘플을 수집 하 고 동물 실험에 참가 했다. 제, 엑스-상대습도, K. 무정 지형 원고 준비에 기여.

프로토콜 및 골 수 유래 NK 분화 세포 (BM-NK)에 대 한 이전 게시 방법20,,2122에 기반. 쥐와 모든 절차는 여는 동물 윤리 실험 위원회 (AEEC) 홍콩의 중국 대학에 승인 되었습니다.
1입니다. 준비 OP9 조건부 매체의
<ol><li>알파-MEM 20 %FBS, 100 U/mL 페니실린 G 및 공기/CO2 의 습도 분위기에서 37 ° C에서 100 µ g/mL 스 murine 기질 세포 라인 OP9 문화 (95%: 5%).<ol>
<li>Hemocytometer, 다음 씨앗 2 x 106 OP9 각 75 cm2 문화 플라스 크에 세포의 중간의 15 mL와 함께 셀을 계산 합니다. 48 h에 대 한 문화입니다.</li>
		<li>문화에는 80% 합류에 도달 하면, PBS의 10 mL로 플라스 크를 씻고 고 플라스 크 당 (FBS와 항생제) 없이 일반 알파 가상 매체의 20 mL를 추가 합니다.</li>
	</ol>
</li>
	<li>24 h 문화 플라스 크에서 OP9 조건 매체를 수집 하 고 0.25 µ m polyethersulfone (PES) 필터 셀 파편 청소 4 ° C (사용 2 주 이내)에서 보존.</li>
</ol>2입니다. 마우스 골 수 세포의 고립
<ol><li>Pentobarbitone 과다 (100 mg/kg, 복 주사)와 함께 12 주 된 C57BL/6J 마우스 안락사 호흡의 부족에 의해 죽음을 확인 다음 뼈 사이 접속점에 수술 칼 커팅으로 대 퇴 골 뼈를 수확 하 고 부드러운 긁 적 하 여 블레이드와 나머지 근육을 제거 합니다.</li>
	<li>냉장된 멸 균 알파-MEM를 포함 하는 50 mL 원심 분리기 튜브에 뼈를 놓고 biosafety 내각에서 다음 단계를 수행 합니다.</li>
	<li>알파-MEM 매체를 삭제 하 고 30 대 70% 에탄올과 뼈를 씻어 s.</li>
	<li>두 번을 나머지 에탄올 차가운 살 균 PBS 가진 뼈를 씻어.</li>
	<li>얼음 처럼 차가운 PBS의 5 mL를 포함 하는 박격포에서 뼈를 전송 하 고는 유 봉과 뼈를 부드럽게 fragmentize (할 하지 격파 그들).</li>
	<li>교 반 하십시오 뼈 부드럽게 골 수 세포는 PBS에 풀어 유 봉 소용돌이 의해. 새로운 50 mL 원심 분리기 튜브로 PBS 포함 하 골 수 세포를 수집 합니다.</li>
	<li>뼈 조각 단단한 흰색 색상에 될 때까지 4 번 2.6에 대 한 단계를 반복 합니다.</li>
	<li>465 x g 4 ° C에서 5 분 동안에 관을 원심 다음 삭제는 상쾌한.</li>
	<li>18 mL의 멸 균 증류수와 펠 릿을 resuspend 하 고 그것은 30 서 적혈구를 제거 하는 s.</li>
	<li>얼음 처럼 차가운 10의 2 개 mL를 추가 X PBS는 반응 중지 하는 다음 lysed 적혈구를 제거 하려면 70 µ m 셀 스 트레이너를 통해 혼합물을 필터링.</li>
	<li>465 x g 4 ° c.에서 5 분 동안에 관을 원심 얼음 처럼 차가운 PBS의 20 mL와 펠 릿 resuspend</li>
	<li>2.11 단계를 두 번 반복 하 여 세포를 씻어.</li>
	<li>100 m m 멸 균 페 트리 접시에 단계의 1.2 OP9 조건부 매체의 10 mL와 20로 보충 셀 펠 릿을 전송 %FBS 0.5 ng/mL의 혼합물 murine 일리노이-7, 30 ng/mL 마우스 SCF와 100 U/mL murine flt3L.</li>
	<li>품 어 2 h. 전송에 대 한 5% CO2 와 37 ° C에서 접시 첨부 되지 않은 셀 5 x 105 가능한 셀/mL (trypan 블루 제외과 hemocytometer 여 수 세포)의 밀도에 새로운 문화 컨테이너로 단계 2.13 에서처럼 동일한 중간 수식 5% CO2와 37 ° C에서 품 어와.</li>
	<li>하루에 4, 변경 OP9 조건부 매체 문화 상태 20 %FBS 및 2000 U/mL murine 일리노이-2의 보충.</li>
	<li>새로 문화 매체 매 3 일; NK 세포로 성숙 7 일 여 하 고 섹션 4에서 자격을 갖춘 수 있습니다.</li>
</ol>3. NK 세포 분화의 유전자 Silencing siRNA 중재
<ol><li>제품 설명서에 따라 transfection 에이전트와 siRNA 또는 넌센스 제어 (NC) 공기로</li>
	<li>0부터 시간 언제 든 지 3.1 단계를 수행 합니다.</li>
	<li>추가 50 nM siRNA 또는 단계 2.14에서에서 셀에 NC 혼합물의.</li>
	<li>실험 끝 지점까지 모든 중간 다과 (예를 들어, 0, 4, 및 7 일)에 3.1 단계를 반복 합니다.</li>
</ol>4. NK 세포 분화 Cytometry 사용의 분석
<ol><li>적절 한 시간에서 세포 분화의 정지를 수집 하 고 얼음 처럼 차가운 PBS로 세척.</li>
	<li>제품 설명서에 따라 셀 고정 버퍼와 셀을 수정 합니다.</li>
	<li>얼음 처럼 차가운 PBS 가진 고정된 셀을 세척 하 고 resuspend Flow Cytometry 얼룩 버퍼 포함 Cy3 활용 된 반대로 마우스 NKp46의 100 µ L에 1 x 106 셀과 1: 100 희석 비율에 반대로 마우스 CD244 항 체 PE 활용.</li>
	<li>샘플 2 h에 대 한 실 온에서 어둠 속에 얼룩.</li>
	<li>Resuspend, PBS의 300 µ L로 얼룩진된 샘플 다음 FACS 데이터 및 Cytobank 플랫폼20 (http://cytobank.org/)와 분석.</li>
</ol>

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저자는 공개 없다.

현재 작업에서 우리는 murine NK 세포 골 수 파생 에 시험관을 생산 하는 새로운 방법을 설명 했습니다. 원래 시스템21,22 에 셀 피더 성공적으로 크게 차별화 시스템의 안정성을 증가 OP9 셀의 조건부 매체에 의해 대체 됩니다. 또한, 시스템 높은 수량을 생산할 수 있는 고 순도 성숙한 NK의 세포 생체 외에서 뿐 아니라 대 한 vivo에서 분석 ...

암 진행은 종양 microenvironment1,2, 호스트에서 파생 된 immunocytes, 예를 들면, NK 세포에 크게 의존 합니다. 여러 학문의 intratumoral NK 세포는 종양 진행3,4부정적인 상관 관계를 설명 했다. 또한, 임상 연구 보여주었다 NK 세포 입양 치료 암5,6,7,,89에 대 한 가능한 전략 이다. NK 세포 기반 암 immunotherapy 최근 단단한 종양을 위한 치료 선택권으로 제안 되었다 하지만 면역 억제 cytokines의 분 비와 고체 종양 의 microenvironment에 ligands 활성화 downregulation 존재 하는 과제 10,11. 변형 시키는 성장 인자-β (TGF-β) 발암에 진압 역할을 제안 하고있다 하지만 역설적으로 암 세포는 또한 TGF-β1 종양 개발12,,1314 를 지원 하기 위해 생산 , 15. 인터페론 응답 다운 규제 및 CD16-중재 인터페론-감마 (IFN-γ) 생산 체 외에서16,17를 통해 NK 세포의 cytolytic 활동을 억제 수 있습니다 TGF-β 신호 18.
TGF-β 신호 종양 microenvironment에의 중단 될 수 있지만 암 제거 가능한 방법, 완전히 TGF-β 신호를 차단 하면 항 염증 기능을 인해 자가 면역 질환의 개발에 의해 입증 부작용 등 조직의 염증, 심장 결함, 그리고 마우스에 면역19모델. 따라서, TGF-β-중재 면역 억제의 작동 메커니즘을 이해 이어질 것입니다 암 치료에 대 한 접근 치료 대상의 식별.
NK 세포 개발에 필요한 분자 이벤트 명료 하 윌리엄스 외 20세포 NK로 murine 골 수 조 혈 줄기 세포 분화에 대 한 생체 외에서 시스템 설립. 이 시스템은 크게 NK 세포21의 소설 창시자의 식별을 포함 하 여 NK 세포 개발의 기계 론 적인 연구를 촉진 한다. 그러나, 골 수 창시자로 피더 레이어20,21, OP9 stromal 세포를 지 원하는 시스템에서 경작 한다 그리고이 다른 유형의 세포 인구는 주로 유전자 방해의 추가 응용 프로그램 제한 도구 (예를 들어, 중재 하는 siRNA 유전자 침묵) 차별화 NK 세포에 구체적으로 적용.
여기, 우리 더 윌리엄스 외20의 생체 외에서 시스템을 수정 하 여 개발 된 피더 무료 시스템을 설명 합니다. 우리의 시스템에 OP9 stromal 피더 세포는 필요 하 고 대신 조건부 매체 최근 NK 세포에 생체 외에서그리고이의 차별화를 영향을 주지 않고 사용 OP9 TGF-β는 암 진행을 통해 홍보할 수 밝히기 위해서 우리를 끌 종양 microenvironment22에 E4bp4 종속 NK 세포 개발 억제 이 새로운 시스템 성공적으로 elucidating 특정 조건 하에서 NK 세포 개발의 분자 메커니즘에 대 한 배경 없는 메서드를 제공 합니다 (예를 들어, 높은 TGF-β1, 중재 하는 siRNA 유전자 침묵, 등.) 생체 외에서.

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			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
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			<td>Gibco</td>
			<td>22561021</td>
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			<td>Gibco</td>
			<td>10500064</td>
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			<td>Gibco</td>
			<td>10010049</td>
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			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
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			<td>PEPROTECH</td>
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			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
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			<td>PEPROTECH</td>
			<td>212-12</td>
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			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
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			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
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			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
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			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
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			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
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a novel feeder-free system for mass production of murine natural killer cells in vitro

자연적인 살인자 (NK) 세포는 타고 난 면역 계통에 속해 있으며 첫 번째 라인 항 암 면역 방어; 그러나, 그들은 종양 microenvironment에서 억제 되 고 기본 메커니즘은 아직 크게 알려진. NK 세포의 일관 되 고 신뢰할 수 있는 소스의 부족 NK 세포 면역의 연구 진행을 제한합니다. 여기, 우리가 시험관에서 시스템 높은 품질과 급지대 무료 조건 murine NK 세포 골 수 유래의 수량을 제공할 수 있는 보고 합니다. 더 중요 한 것은, 우리는 또한 그 중재 하는 siRNA 유전자 침묵이 시스템을 사용 하 여 E4bp4 종속 NK 세포 성숙 억제 성공적으로 보여 줍니다. 따라서,이 소설 체 외에서 NK 세포 분화 시스템 면역 연구를 위한 소재 솔루션입니다.

대표 결과 설명된 프로토콜에 따라 얻을 수 있습니다. 총 골 현 탁 액 셀 피더 무료 차별화 시스템에서 11 일; 재배 했다 확산 속도에 상당한 증가 하루 7 일 0 (그림 1A)에 총 셀 개수와 비교 하 여 관찰 되었다. 높은 세포질에 핵 비율 및과 립 풍부한 세포질 형태학 NK 세포로 성숙 시스템 (그림 1B)에서 6 일?...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

여기, 선물이 murine NK 세포 유전자 침묵 시키기 기계 연구 생체 외 및 생체 내에서피더 무료 차별화 시스템을 사용 하 여 대량 생산 하는 프로토콜.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

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Research

JoVE Journal

Immunology and Infection

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

7.1K 조회수.  The Chinese University of Hong Kong. 여기, 선물이 murine NK 세포 유전자 침묵 시키기 기계 연구 생체 외 및 생체 내에서피더 무료 차별화 시스템을 사용 하 여 대량 생산 하는 프로토콜.

비디오: A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

연구

면역학 및 감염병학

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

인공 항원은 세포 (aAPC) 중재의 활성화와 자연 킬러 T 세포의 확장을 제시

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

유리 - 지원 평면 지질 이중층에 자연 살해 세포 면역 시냅스의 슈퍼 해상도 이미징

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

마우스 비장에서 불변 자연 살해 T 세포를 분리하고 확장을위한 최적화 방법

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

랫 트 태 반 천연 킬러 세포 세포 용 분해 기능의 평가를 위한 플레이트 기반 세포 독성 분석

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

치료 항체의 존재에 FcγRIIIa 참여에 의해 구동 인간 자연 킬러 세포 이벤트의 평가

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

인간 자연 킬러 세포 대사의 분석

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...