이 프로젝트는 국민 건강 기금 HL087088 (MK)의 연구소와 HL18208 (MK)에 의해 지원되었다.

1. 인간의 T Lymphocytes의 분리 <ol><li> 건강한 기증자로부터 인간의 혈액을 구합니다. 혈액은 다음 단계로 진행하기 전에 실내 온도 (~ 30 분)로 냉각 수 있습니다. </li><li> 부드럽게 8 ML 왕복 하단의 폴리스티렌 튜브로 실내 온도 Polymorph 밀도 기울기 미디어 3 ML을 피펫. 부드럽게 Polymorph 미디어 위에 전체 혈액의 3 ML를 추가합니다. 두 시약의 혼합되지 않도록하는 것이 중요합니다. </li><li> 실온에서 45 분간 500 XG에서 튜브를 원심 분리기. </li><li> 원심 분리에 따라, 말초 혈액 mononuclear 세포 (PBMC)는 이제 최고 셀 레이어에 다른 혈액 구성 요소에서 분리합니다. PBMC 계층은 첫 번째 흐림 밴드로, 위에서 아래로 나타납니다. </li><li> 조심스럽게 PBMC 레이어 위에 혈액의 맑은 노란색 상위 단계를 제거하고 15 ML 50 ML 원뿔 관에 PBMC 레이어를 전송하는 P1000 micropipette를 사용합니다. </li><li> 오분 때마다 500 XG에서 세포를 centrifuging, PBS로 두 번 PBMC 씻으십시오. 뜨는은 각 씻고 나서 약간 뿌연됩니다. </li></ol> 2. 인간의 T Lymphocytes의 문화 <ol><li> 피펫 사용하여 10 % FBS를 포함한 20 ML RPMI 1640 미디어 T - 75 문화 플라스크에 PBMC을 전송, 1 % 페니실린 / 스트렙토 마이신, 1 μg / ML phytohemagglutinin (PHA). </li><li> 37 품어 ° C와 5% 적어도 1 시간 및 최대 24 시간 동안 CO 2. 이 단계는 중단에 남아 lymphocytes에서 분리된 것으로, 술병의 표면에 점착 성의 것입니다 monocytes을 수 있습니다. 짧은 부화 (1 시간)가이 단계에서 사용하는 경우, 그것은 단계 2.1에 명시된 바와 같이 PHA와 보완없이 10% FBS, 1 % 페니실린 / 스트렙토 마이신을 포함하는 RPMI 1640 미디어를 사용하기 위해 허용됩니다. </li><li> 신중하게, 술병에서 미디어를 모두 제거 50 ML 원뿔 관에 추가하고, 5 분 500 XG에 원심 분리기. </li><li> 지금은 주로 lymphocytes을 포함하는 세포 펠렛을 Resuspend, 10 % FBS를 포함한 25 ML RPMI 1640 미디어를 포함하는 새로운 T - 75 플라스크에 세포를 전송, 1 % 페니실린 / 스트렙토 마이신, 1 μg / ML PHA. </li><li> 삼일 (PBMC의 초기 부화가 야간 있었다면 이일)에 대한 37 ° C에서 알을 품다. 성장 후 24 시간 후, 신선한 미디어 15-20 ML을 추가하고 큰 T - 175 플라스크로 전송해야 할 수도 있습니다. </li><li> 삼일 후, 50 ML 원뿔 관에 술병과 전송에서 미디어 및 정지 lymphocytes를 제​​거하는 피펫을 사용합니다. 5 분 500 XG에 원심 분리기. </li><li> 10% FBS 25 ML (T - 75) 50 ML (T - 175) RPMI 1640을 포함하는 새로운 T - 75 또는 T - 175 플라스크에 세포 펠릿 및 전송 세포를 Resuspend, 1 % 페니실린 / 스트렙토 마이신, 20 NG / ML 인간 IL - 2 또는 IL - 15. </li><li> 4-7일에 대한 lymphocytes 성장. T - 75 플라스크로 시작하는 경우, 문화 1-2 일 후에 확대와 T - 175 플라스크로 전송해야합니다. </li></ol> 3. 관내 림프구 마이 그 레이션 분석에서 <ol><li> 마이 그 레이션 분석하기 전에 일일, 코트 4 PBS에서 A 또는 G 하루 20 μg / ML 단백질과 유리 바닥 0.17 mm 요리 ° C. </li><li> PBS로 광범위하게 접시를 씻으십시오. </li><li> 인간 ICAM-1/Fc (10 μg / ML)과 인간 SDF - 1 접시에 PBS 용액에서 이러한 시약을 추가하고 실온에서 4 시간 잠복기 (2 μg / ML).를 무력화 </li><li> 먼저 hemacytometer를 사용하여 T - 175 플라스크에 문화의 세포 밀도를 결정하여 T lymphocytes를 준비합니다. 약 2-5 X 10 5 세포는 마이 그 레이션 분석을 위해 적절한 세포 밀도를 달성하기 위해 요리마다 사용해야합니다. </li><li> PBS로 두 번 T lymphocytes 씻으 다음 1 MG / ML D - 포도당을 포함하는 하나 ML L - 15 미디어 resuspend. </li><li> 광범위하게 PBS로 ICAM-1/Fc와 SDF - 1 코팅 접시를 씻으십시오. </li><li> 접시 1 ML 미디어에서 T의 lymphocytes을 전송하고 37 그들을 유지 ° C. </li></ol> 4. NIS 요소 소프트웨어를 사용하여 이미지 시퀀스를 캡처 <ol><li> NIS 요소 소프트웨어를 여십시오. </li><li> 카메라 설정 메뉴에서, 살아있는 영상 및 이미지 캡처 모두 모드로 &quot;2x2 비닝&quot;을 선택합니다. </li><li> 응용 프로그램 메뉴로 가서 실행 / 정의 실험을 선택합니다. </li><li> 이미지와 이미지 캡처 시퀀스의 총 길이 사이의 시간의 길이를 선택합니다. </li><li> 이미지 수집을 시작하기 위해 &quot;실행&quot;버튼을 누르십시오. </li><li> 전지 추적할 수 및 마이 그 레이션 매개 변수 (속도, 경로 길이, 변위 등) ImageJ, AutoQuant 또는 Volocity (그림 1)를 포함한 여러 소프트웨어 패키지 중 하나를 사용하여 계량. </li><li> 각각의 셀 및 timepoint 수집 (그림 2)와 원점에서 각 셀에 대해 일반적인 시작 지점 (그림 3)과 그래프에 예상이됩니다 생성하려면 &quot;거미줄 계획을,&quot;XY 좌표. </li></ol> 5. 대표 결과 IL - 2 또는 IL - 15, T의 lymphocytes 중 하나의 존재에 문화의 일 6뿐만 아니라 긍정적인 CD4 및 / 또는 CD8 얼룩 같은 긍정적인 인 경우에는 3 번 CD의 얼룩에 의해 결정 세포의&gt; 98%을 구성합니다. IL - 2에 대해서는 83 % CD4 + 세포가 15 % CD8 +와 &lt;1 %의 CD4 + CD8 + 세포를 발견했습니다. IL - 15의 경우, 우리는 88 %의 CD4 + 세포, CD8 + 11% 및 &lt;1 %의 CD4 + CD8 + 세포를 발견했습니다. ICAM-1/SDF-1 기판에 대한 T 림프구 마이 그 레이션하는 동안, 세포는 1 시간 기간 동안 유지할 수 약 15 μm의 / 분 속도를 전시. ICAM-1/SDF-1에 대한 T 림프구 마이 그 레이션, LFA - 1 - 중재 접착에 따라 달라집니다 같은 반 LFA - 1 리간드 차단 항체 것은 심각하게 마이 그 레이션을 억제. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="그림 1" /> 그림 1. T lymphocytes 마이 그 레이션의 세포 추적. 전체 혈액에서 분리된 6 일간 IL - 2 또는 IL - 15의 면전에서 교양 T의 lymphocytes 10 μg / ML ICAM - 1과 함께 코팅 유리 바닥으로 요리를 준수하고 마이 그 레이션하는 허용되었습니다 2 μg / ML SDF - 1. 이미지는 30 분 10 초마다을 인수했다. 세포는 각 이미지를 식별하고 Volocity 소프트웨어를 사용하여 시간이 지남에 추적되었다. 이 영화는 ~ 15 μm의 / 분 속도로 T lymphocytes의 임의의 마이 그 레이션을 보여줍니다. <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">비디오를 보려면 여기를 클릭하십시오.</a> T lymphocytes 마이 그 레이션의 영화 1. 세포 추적. T의 lymphocytes는 전체 혈액에서 분리의 면전에서 교양 IL - 2 6 일 또는 IL - 15는 10 μg / ML ICAM - 1, 2 μg / ML SDF - 1 코팅 유리 바닥으로 요리를 준수하고 마이 그 레이션하는 허용되었습니다 . 이미지는 30 분 10 초마다을 인수했다. 세포는 각 이미지를 식별하고 Volocity 소프트웨어를 사용하여 시간이 지남에 추적되었다. 이 영화는 ~ 15 μm의 / 분 속도로 T lymphocytes의 임의의 마이 그 레이션을 보여줍니다. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="그림 2" /> 그림 2. Spatiotemporal 세포 위치. 각 세포의 XY 좌표는 Volocity 소프트웨어를 사용하여 각 시점에 대한 취득했다. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="그림 3" /> 그림 3. &quot;스파이더 웹 플롯.&quot; XYt를 사용하면 각 셀에 대한 15 세포가 무작위로 선택되었으며 원점에서 일반적인 출발점와 제휴해서 역모를 조정합니다. 거미 웹 음모를 비교하면 서로 다른 실험 조건 사이의 마이 그 레이션의 차이를 빠르게 시각적으로 묘사를 제공합니다. 제어 조건 하에서 T 림프구 마이 그 레이션 (왼쪽 패널) 및 안티 LFA - 1 리간드 차단 항체 (오른쪽 패널)의 존재에 플롯.

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

본 실험에서는, 우리는 기본 인간 T의 lymphocytes의 운동성를 분석하는 간단한 시스템에 대한 자세한 내용을 제공합니다. 체외 마이 그 레이션 assays 다양한 세포 유형의 운동에 관련된 다양한 분자와 신호 경로의 역할을 해부하다하는 데 사용되었습니다. 우리 프로토콜에서 자신의 실험을 설계할 때 염두에 두어야하는 몇 가지 중요한 컨트롤은 다음과 같습니다 : 1) 소 혈청 알부민 (BSA)...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Remel/Fisher Scientific</td>
<td>R30852801</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>202-IL</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-15</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>247-IL</td>
<td>&nbsp;</td>
<tr>
<td>Protein G</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human SDF-1</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>350-NS</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

human t lymphocyte isolation, culture and analysis of migration in vitro

T lymphocytes의 마이 그 레이션은 다른 세포 또는 세포외 기질 단백질로 표현 리간드와 함께 세포 표면 integrins의 접착제 상호 작용을 포함한다. 낮은 친화 상태에서 세포 첨단에 높은 친화력 상태로 integrins의 정확한 spatiotemporal 활성화 T 림프구 마이 그 레이션에 대한 중요<sup&gt; 1</sup&gt;. 마찬가지로, 세포 후행 가장자리의 철회는, 또는 uropod, 영구적인 인테 종속적인 T 림프구의의 운동성을 유지하는 데 필요한 단계입니다<sup&gt; 2</sup&gt;. 수단으로 면역 또는 염증 질환 대상 integrins 많은 치료 방법은 leukocytes의 과도한 모집 및 마이 그 레이션을 억제하기 위해<sup&gt; 3</sup&gt;. 인간의 T 림프구의 마이 그 레이션을 조절 분자 이벤트를 연구하기 위해, 우리는를 활용해야<em&gt; 체외에서</emT 림프구는 vasculature에서 모집하는 동안 발생하는 환경을 모방 2 차원 기판의 셀 마이 그 레이션을 분석&gt; 시스템. T의 lymphocytes 먼저 인간의 기증자로부터 격리되고 나서 자극을 7 일에서 열흘 동안 양식입니다. 분석하는 동안, T의 lymphocytes는 세포 유착 분자 -1 (ICAM - 1), 인테 LFA - 1에 대한 리간드와 stromal 세포 - 파생 요소 - 1 (SDF - 1) 코팅 기판에 준수 및 마이 그 레이션하는 허용됩니다. 우리의 데이터는 T lymphocytes는 ~ 15 μm의 / 분 철새 속도를 전시 것을 보여줍니다. T 림프구 마이 그 레이션은 인테 그린의 봉쇄에 의해 저해 수 있습니다<sup&gt; 1</sup&gt; 또는 세포 actomyosin 기계 억제제에 의해 그 세포 마이 그 레이션을 조절<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T 림프구 마이 그 레이션 림프 기관, vasculature에서 출구로 유도하는 동안 발생하고, 주변 조직을 체결. 여기, 우리는 T 림프구 마이 그 레이션을 분석하는 데 사용할 수있는 프로토콜을 설명<em&gt; 체외에서</em&gt;.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

46.1K 조회수.  University of Rochester. T 림프구 마이 그 레이션 림프 기관, vasculature에서 출구로 유도하는 동안 발생하고, 주변 조직을 체결. 여기, 우리는 T 림프구 마이 그 레이션을 분석하는 데 사용할 수있는 프로토콜을 설명 체외에서.

비디오: Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

기본 인간 T 세포 piggyBac Transposon 시스템 변경

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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면역학 및 감염병학

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

표현형 분석 및 대한 인간의 피부에서 림프구 분리<em&gt; 전의 VIVO</em&gt; 세포 배양

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

마우스 나이브 CD4<sup&gt; +</sup&gt; T 세포의 분리 및<em&gt; 시험관</em&gt; T 세포 하위 집합으로 차별화

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

염증이 진피의 이미징 CD4 T 세포 간질 마이그레이션

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

인간 Monocyte Chemotaxis에서 생체 외에서 그리고 Vivo에서 인신 매매 Murine 림프 구 측정

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

분석 Murine 가장자리 지역 B 세포 생체 외에서의 전단 흐름 유도 마이그레이션

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...