Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

기본 백혈병 세포 1. 호중구의 분리 및 공동 문화 참고 : 절차는 동의서에 서명 모든 환자와 리옹 병원 윤리위원회의 승인하에 실시 하였다. <ol><li> 기본 백혈병 세포 및 호중구의 분리 <ol><li> 만성 림프 구성 백혈병 (CLL)으로 진단 된 환자에서 EDTA (혈액의 밀리리터 당 1.8 mg을 EDTA)에 말초 혈액의 튜브를 수집합니다. </li><li> 멸균 50 ㎖ 튜브에 혈액을 각각 15 ㎖에 ?...

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밀도 구배 원심 분리 방법을 이용하여 고순도의 인간 혈액 호중구의 분리를 위해 동일한 단계 단핵 세포 내에서, 여기에 효과적인 간단한 빠르고 저렴 프로토콜 설명도 분리 회수된다. 분리 된 세포 집단은 ≥90 % 순수 있습니다. 여러 가지 방법 인간의 혈액에서 호중구의 분리에 사용할 수 있습니다. 이러한 불연속 그라디언트 (11, 12)를 사용하거나 호중구는 면역 자?...

선천성 면역 세포는 종양 미세 환경 내에서 세포의 중요한 비율을 구성하는 환자, 암 (1)의 동물 모델에서 종양의 암과 연관되어왔다. 최근에,보다 널리 만성 면역 반응은 화학 요법에 대한 종양이 진행, 전이 저항 증진에 중요한 역할을하는 것으로 인식되고있다. 식세포 직접 화학 요법에 3,4- 종양 세포 반응을 조절하는 것으로 나타났다 중요한 선천성 면역 세포이다. 그러나 항암 치료에 대한 종양 반응을 조절하는 호중구의 선천 면역 키 선수의 역할은 알려져 있지 않다. 이러한 프로토콜의 목적은 CLL 환자의 혈액 샘플로부터의 호중구를 분리하는 빠르고 신뢰할만한 방법을 사용하여 안티 림프종 에이전트 림프종 세포의 민감도를 조절하는 역할을 연구하기 위하여 과립 통로 따라 HL60 세포를 구별한다. 6 침입에 대한 방어의 첫 번째 줄 혈액 (5) 행동의 선천성 면역 시스템의 가장 풍부한 세포의 구성 요소입니다. 호중구 여러 병리 적 상태 (7)에 가변 이펙터 기능 이외에 유효 선천성 면역 반응을 상승에 필수적인 역할을한다. 따라서, 이러한 농도 구배 분리법과 같은 다른 혈액 세포에서 호중구를 분리하는 빠르고 신뢰할만한 방법은 시험 관내 연구에 필요하다. 호중구 격리를 위해이 방법을 사용하면 생체 내 및 생체에서 호중구 매개 면역 기능에 대한 추가 연구를 촉진 할 것이다. 호중구 순수한 집단을 수득하는 능력은 면역 학적 질환 환자의 팔 조사 중요한 첫 단계이다. 밀도 구배 분리 방법은 세포의 높은 수율을 수득하는 이상적인 방법이다. 운전 방식D는 쉬지 않고 35 분 동안 300g으로 원심 분리 한 다음 희석 된 인간 혈액을 함유하는 튜브의 바닥에서 밀도 구배 용액의 첨가를 포함한다. 단핵 세포의 링 인터페이스에 표시하고, 호중구 전 이하 상주. 이 방법은 훨씬 더 9 비싼 호중구 분리 키트로 사용 가능한 다른 방법에 대한 상당한 이점이있다. 또한, 인간의 호중구에 대한 특정 표면 마커에 관한 항체를 사용하여 상업용 키트에 의해 인간 혈액으로부터 분리 호중구 세포 활성화 또는 분화의 위험을 증가시킨다. 밀도 구배 분리 방법은 단시간 호중구의 분리를 허용한다. 동일 단계 내에서 단핵 세포는 분리 회수된다. 순수한 세포의 높은 수율 무결성 기능을 달성하기 위해 수득되는 기본 기술이다. 종양 microenv을 모방하기 위해ironment, 3 차원 실험을 수행 하였다. 체외에서 호중구의 짧은 반감기를 고려할 때, 신선한 인간의 호중구와 3D 실험은 결정적인 없습니다. 이런 이유로, 전 골수성 (HL60) 세포 분화 유도제 디메틸 설폭 사이드 (DMSO) 및 레티노 산 (RA)를 사용하여 호중구와 같은 세포로 분화 유도된다. 차별화 된 HL60 세포 (HL60은 diff)를 사용하여 인해 다른 기증자로부터 격리에 호중구의 다른 응답을 갖는 것을 방지 할 수 있습니다. 체외 차원에서 문화 모델은 체외 2D 모델 사이의 생체 내 모델에서 중간 단계를 나타냅니다. 2D 문화에서, 세포는이 합성 표면에 변성되어 퇴적 단백질에 자연스러운 세포의 첨부 파일을 형성하는 플라스틱 표면에 확산. 반대로, 세포가 세포 외 기질의 합성 보낸 3D 배양 형태 천연 세포 - 세포 부착에 세포는 이들이 결합되어있는 천연 물질이다.이러한 이유로, 특히 암 세포 및 다른 세포 유형 간의 3D 공동 배양 모델은 종양 성장, 혈관 신생 및 전이에 대한 기여를 나타내는 매우 유용한왔다. 결과적으로, 3 차원 배양은 세포 배양 물이 생체 내 (10)에 존재하는 생리적 조건을 모방 할.

<table border="0" cellpadding="0" cellspacing="0" width="1161">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">RPMI 1640&#160;</td>
			<td style="width:157px;">Gibco Invitrogen</td>
			<td style="width:156px;">21875-034</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td>
			<td>Life technologies</td>
			<td style="width:156px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin (Pen Strep)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruton&#39;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&#160;</td>
			<td style="width:157px;">CliniSciences</td>
			<td style="width:156px;">A3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine&#160;</td>
			<td style="width:157px;">EG labo</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Red cell lysis buffer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:156px;">555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll (Pancoll)</td>
			<td>PAN Biotech</td>
			<td style="width:156px;">P04-60500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Retinoic acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine serum albumin (BSA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>A7906</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">E5134</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium chloride (NaCl)</td>
			<td>Euromedex</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexing V-FLOUS staining kit&#160;</td>
			<td style="width:157px;">Roche</td>
			<td>11&#160;988&#160;549&#160;001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kit RAL 555 Modified Giemsa staining kit</td>
			<td style="width:157px;">Cosmos Biomedical</td>
			<td style="width:156px;">CB361550-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cytocentrifuge</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMR-XA microscope</td>
			<td style="width:157px;">Leica Microsystems</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cellometer Auto T4 Cell Viability Counter</td>
			<td style="width:157px;">Nexcelom&#160; Bioscience</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

흰색 환 단핵 세포이고, 혈소판 혈장 :. 여기에 설명 된 농도 구배 분리법은 CLL 환자의 혈액에서 분리 된 일차 백혈병 세포 및 자극되지 호중구도 1a는 위에서 아래로 (밀도 구배 원심 분리 후의 다른 혈액 층을 나타낸다 제공 밀도 구배 용액, 과립구 및 적혈구).도 1b 및 1c는 각각 호중구 (멀티 로브 핵 세포)와 단핵 세포의 형태 학적 외관의 차이를 ?...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Research

JoVE Journal

Immunology and Infection

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

흐름 조건에서 인간 제대 정맥 내피 세포 및 호중구 윤회의 연구에서 이들의 사용 격리

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

연구

면역학 및 감염병학

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

인간의 두 그라데이션 원심 분리하여 단핵구와 테프론 코팅 세포 배양 가방에서 대 식세포로 분화의 분리

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

방법은 세균 Pyomelanin 생산을 억제하고 산화 스트레스에 감도에 대응 증가를 확인하는 방법

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

체외 방법에서 대상 바인딩 및 치료 항체 사이 CDC 유도 비교 : 응용 프로그램 생물학적 동등성 분석에

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

조직학 정량화 폐 실험 Aspergillosis에 곰 팡이 부담을 결정 하

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

생체 외에서 형광 현미경 검사 법에 의해 개 호 중구 Extracellular 함정 형성: 역동적이 고 양적 분석

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

예방 접종 후 기니 돼지 모델에서 측정 T 세포 응답에 최적화 된 인터페론-감마 ELISpot 분석 결과

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

멀티플렉스 형광 Immunohistochemical 얼룩, 이미징, 및 림프 종 조직학 샘플 분석

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...