We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

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YK, ILG, TLM, WLM, DPH 및 KSB : 없음 경쟁 금전적 이해. ANT는 베크만 쿨터의 직원이며,이 비디오 문서의 주식 공개는 벡크 만 쿨터 후원

말초 혈액의 면역 표현형은 면역 개별 응답에 대한 통찰력을 확보하기위한 매우 중요합니다. 문제는 실험 - 투 - 실험 변동성 1,14을 제어하는 분석의 표준화에있다. 변화 중 하나는 주요 소스 샘플의 인간의 조작에있다. 따라서, 시료 처리의 부분 또는 전체 자동화 실험 간 실험 가변성 1,14 극적인 감소를 용이하게 생각할 수있다. 이 프로토콜에서는, 우리는 전체 혈액의 면역 표현형의 샘플 염색에 대한 2 차원 바코드 판독기가 장착 된 자동화 액체 핸들러를 도입하여 분석 변동성을 최소화하기 위해 성공적인 노력을보고한다. 디자인, 우리의 방법은 다양합니다 그들을 정의하는 추가 &quot;자료 칵테일&quot;을 추가하여 다른 면역 하위 집합을 모니터링 할 수 있습니다. 이러한 서브 세트들은 T 헬퍼 세포, T 조절 세포, B 세포, NK 세포, 수지상 세포 및 단핵구 (원고를 포함준비) 18. 우리는 추가 마커 1을 도입하여 컨소시엄의 추천 항체 패널을 채택. 우리가 유도 항원의 검출에 대한 이러한 채널을 예약하고 싶었다로 세포 집단은, 검출기 화려한 보라색 421 (BV421)에 최소한의 방출과 형광 색소에 접합 된 &quot;자료 칵테일&quot;, 피코 에리 트린 (PE)를 사용하여 식별 및 알로 (APC) 하였다. 이 기능은 면역 세포의 활성화 상태를 모니터링 할 수있는 가장 높은 감도를 보장뿐만 아니라,이 세 가지 형광 색소가 가장 많이 유도 마커에 대한 항체가 결합 될 때 새로운 활성화 마커를 유연하게 숙박 할 수 있습니다뿐만 아닙니다. 따라서, 우리의 방법뿐만 아니라 전체 혈액의 면역 표현형을위한 칵테일 제조에 적합뿐만 아니라 세분화 된 조직 (예를 들면, 종양)에서 발견 한 말초 혈액 단핵 세포와 세포를 포함하는 다른 샘플을 염색하는 데 유용합니다. 성공적인 소개우리의 방법의 duction 특별한 실험 실용 준비의 단계에 대한 관심, 그리고 프로그래밍 및 방법의 실행을 필요로한다. 2 차원 바코드 튜브의 제조는 사람이 읽을 수있는 라벨과 지정된 튜브에 항체의 전송과 라벨을 포함한다. 오류의 도입을 방지하기 위해, 우리는 두 사람과이 작업을 수행하는 것이 좋습니다. 스프레드 시트를 변경 할 때마다, 연구자들은이 방법이 제대로 작동하는지 확인해야합니다. 프로그래밍시 적절한 피펫 팅 방법을 선택하는 것은 성공적인 액체 전송을 보장합니다. LLS는 우리가 P50 또는 P200 전도성 팁을 사용하는 항체 튜브의 바닥에서 침전 된 이물질을받지 않고 피펫 팅을 할 수 있습니다. LLS는 P1000 팁 더 민감하고 때로는 거짓 거품의 존재에 의해 트리거로 LLS는 P1000 팁에 대한 좋은 선택지가되지 않을 수 있습니다. 팁의 가장자리 사이의 공간이 충분하지 않으면 실험 실용 정의의 높이는, 예를 들면, 같은 차선의 액체 전사가 발생할 수있는 각 기기 조정되어야튜브의 바닥. 도 12에 도시 된 바와 같이 &quot;기본 칵테일&quot;및 / 또는 &quot;활성화 칵테일&quot;을 잘 볼 텍싱되지 않은 경우, 그것은 최적의 얼룩을 초래할 수있다. 우리는 시약 (19) 분배와 관련 변동성을 줄이기 위해 다른 방법으로 세포 염색을 위해 동결 건조 된 시약을 사용하는 방법에 대해 생각했다. 그러나, 브릴리언트 바이올렛 염료 고분자 접합체 서로 일으키는 비특이적 신호와 상호 작용하는 것으로 알려져있다. 이와 같이, 동결 건조 된 시약에 두 개 이상의 중합체 접합체 (예 BV421 및 BV650 등) 비 특정 시그널링이 발생할 수 첨가 (예 BV421 + 인구 BV650 배경 신호의 증가) 20. 또한, 동결 건조 된 시약은 새로운 염색 등의 유연성이 부족하다. 그들은 일반적으로 더 비싸고 대량 주문이 필요합니다. 이러한 이유로, 2 차원 바코드 튜브 장착 자동화 액체 핸들러를 사용하여 선택 하였다. 그것은 톤 있지만AKE를 시간을 설정하고 이러한 요인은 생산성 향상과 분석의 재현성에 의해 보상됩니다 장기적으로 악기를 구입하기 위해 선행 투자를 수반합니다. 사실, 몇몇 그룹은 이전에 면역 표현형 또는 유사한 애플리케이션 (21, 22)의 워크 플로우에 자동화 액체 핸들러의 성공적인 통합을보고했다. 유동 세포 계측법 분석을위한 자동화 솔루션은 상용 소스 (FACS SPA III, 자동 칵테일 제조 워크 스테이션 및 FlowStainer)에서 사용할 수 있습니다. 이것은 또한 면역 표현형에 대한 자동화 된 칵테일 준비를위한 큰 필요가 있음을 나타냅니다. 이 기술을 마스터 한 후, 우리는 완전 자동화 된 전체 혈액의 면역 표현형의 개발은 더 실험 - 투 - 실험 변동성을 줄일 수, 심지어 23을 설정 다기관 임상 시험에서 전체 혈액의 면역 표현형이 가능 할 수 있다는 구상. 우리는 이미를 Lyse이었다 사용하기 시작했다시간 조수 용해 및 세척 단계를 자동화합니다. 또한 2D 바코드 튜브 항체의 양과 시약 분배 추적 자동 판정 크게 각각 항체의 재고 및 우리의 방법을 통해 품질 관리를 향상시킬 수 있음을 상상할.

인간의 말초 혈액의 면역 표현형은 면역 세포 하위 집합 1 양적, 질적 변화를 결정한다. 이 작업 및 저항 예측 바이오 마커 에이즈 발견 메커니즘에 대한 통찰력을 제공하고, 복합 면역 요법의 개발을 용이하게한다. 따라서, 면역 표현형의 검증 및 표준화 학술 연구자, 임상 실험실과 산업에 상당한 관심의 영역입니다. 유세포 방법에 의해 말초 혈액의 면역 표현형 현재 혈액 학적 악성 종양 3 및 인간 면역 결핍 바이러스 (HIV) 감염 4,5, 면역 요구에 대한 말초 혈액 신뢰성 면역 표현형 그 때문에 검증 및 표준화 할 사항의 임상 관리에 사용되지만 면역 세포 하위 집합 및 활성화 / 억제 수용체 1,6-8보다 광범위한 범위를 필요로한다. Successful 면역 표현형은 장비 설치 및 세포 얼룩 절차 9,10에 관한 실험 - 투 - 실험 변동성을 최소화하기 위해주의 표준화가 필요합니다. 유동 세포 계측법에 대한 장비 설정 표준화가 잘 전 우려 9,11,12를 해결하기 위해 설립 동안, 면역 세포의 서브 세트 및 활성화 상태의 범위를 제한하지 않고 세포의 염색에 관한 변동을 최소화하는 방법을 불분명. 항원의 수가 증가를 검출 할 때, 너무도 오차 변동은 최적의 시약 분주과 교차 오염에 기인 기회를한다. 응고 전혈 phenoptypic 분석 방법 임상 화학 분석에 사용하기 위해 1980 년대 후반에 설립되었다. 이 같은 방법은 샘플 준비를위한 연구 실험실의 요구를 제공하고 있습니다. 중요한 것은, 임상 실험에 사용 된 항체 칵테일은 통상적으로 덜 복잡하다 아바ilable 사전 역가 및 제조 업체에서 미리 혼합. 미리 혼합 된 항체 칵테일의 단 하나의 전송이 필요합니다. 연구 설정에서 10-16 항체의 항체 칵테일 전형적인. 각각의 칵테일 실험실에 의해 안정성을 검증하거나 각 분석하기 전에 신선한 준비해야합니다. 샘플에 대해 여러 항체 칵테일을 준비 50-80 개별 항체의 피펫, 지루하고 오류가 발생하기 쉬운 둘 다 작업을 수반 할 수 있습니다. 칵테일 제조의 자동화는 적은 오류와 같은 수동 칵테일 준비에 비해 여러 가지 장점을 가지고, 피펫의 정확도를 증가하고, 어쩌면 시약 소모 감소했다. 여기서는 제제를 샘플링 관련 변동성을 최소화하기 위해 면역 표현형의 세포 염색 방법으로 2 차원 (2D) 바코드 리더를 구비 한 자동화 된 액체 핸들러의 성공적인 도입을보고한다.

<table border="0" cellpadding="0" cellspacing="0" width="1546">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:613px;">BD LSRFortessa</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:116px;"></td>
			<td style="width:629px;">Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NXp Span-8 Laboratory Automation Workstation&#160;</td>
			<td>Beckman Coulter</td>
			<td>A31839</td>
			<td>Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insert, Tube, 11 mm, White, for 1.5 mL Microfuge Tubes (case of 25)</td>
			<td>Beckman Coulter</td>
			<td>373696</td>
			<td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LLS kit</td>
			<td>Beckman Coulter</td>
			<td>719262</td>
			<td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-Position Tube Rack</td>
			<td>Beckman Coulter</td>
			<td>373661</td>
			<td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOHAZARD AUTOCLAVE BAGS 12x24 IN. RED</td>
			<td>LabMart</td>
			<td>M111416</td>
			<td>Disposable autoclave bags for the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VisionMate High Speed 2D Barcode Reader</td>
			<td>Thermo Scientific</td>
			<td>AB-1850</td>
			<td>2D barcode reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-Channel Handheld Screw Cap Capper/Decapper</td>
			<td>Thermo Scientific</td>
			<td>4105MAT</td>
			<td>For de-capping and capping 2D barcode tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-335</td>
			<td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack XT CFT24</td>
			<td>biocision</td>
			<td>BCS-534</td>
			<td>To hold sample tubes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mL Matrix ScrewTop Amber Tubes in Latch Racks</td>
			<td>Thermo Scientific</td>
			<td>3741AMB</td>
			<td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1025 &#181;L</td>
			<td>Beckman Coulter</td>
			<td>987925</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &#181;L (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>B01091</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>394627</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td>RBC lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Round-Bottom Polystyrene Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-5</td>
			<td>Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD3 Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0038-42</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD4 PerCPCy5.5</td>
			<td>BD Biosciences</td>
			<td>341654</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRgd BV650</td>
			<td>BD Biosciences</td>
			<td>564156</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRab BV786</td>
			<td>BD Biosciences</td>
			<td>563825</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD8 APC-H7</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CCR7 PE-CF594</td>
			<td>BD Biosciences</td>
			<td>562381</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45RA PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>337167</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-HLA-DR BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562804</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD278(ICOS) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557802</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD38 APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">340439</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD279(PD1) BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562516</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD357(GITR) PE</td>
			<td>eBioscience</td>
			<td style="width:116px;">12-5875-42</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD137(41BB) APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">550890</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD69 BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562884</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD314(NKG2D) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557940</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD134(OX40) APC</td>
			<td style="width:188px;">BioLegend</td>
			<td>350008</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td>
			<td>Fisher Scientific</td>
			<td>02-689-6</td>
			<td>For collecting blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td>
			<td>EMD Millipore</td>
			<td>126593</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin, sodium salt</td>
			<td>Affimetrix</td>
			<td>16920</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium, without Phenol Red</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30588.02</td>
			<td>For flow wash buffer</td>
		</tr>
	</tbody>
</table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

흐름에 의한 말초 혈액의 면역 표현형은 세포 계측법 질병과 치료 기간 동안 주변 백혈구의 주파수 및 활성화 상태의 변화를 결정한다. 그것은 치료 효과를 예측하고 새로운 치료 표적을 식별 할 수있는 가능성이있다. 전체 혈액 얼룩은 시료 준비 중에 발생할 수있는 유물을 최소화 조작되지 혈액을 사용합니다. 그러나, 전혈 염색은 샘플의 무결성을 보장하기 위해 신선하게 수집 한 혈액에서 수행되어야한다. 또한,이 탠덤 염료의 잠재적 불안정성을 방지하고 선명한 보라색 염료 시약 사이의 상호 작용을 방지하기 위해 같은 날 항체 칵테일을 제조하는 것이 가장 좋다. 따라서, 전체 혈액 얼룩은 내 간 실험적인 변화를 통제하기 위해주의 표준화가 필요합니다. 여기서는 antibod 제조 표준 과정으로 2 차원 (2D) 바코드 리더를 구비 한 자동화 된 액체 핸들러 배포 신고유동 세포 계측법에 대한 Y 칵테일. 항체는 데이터베이스에서 컴파일 된 96 웰 형식 및 내용에 배치 2D 바코드 튜브에 옮겼다. 액체 핸들러는 데이터베이스 내 항체 이름을 참조하여 소스 항체 튜브를 찾을 수있다. 우리의 방법은, 소스 항체 튜브의 위치 조정을 위해 지루하고 제거. 또한 사용자가 용이하게 분석 용 소프트웨어 방법에서, 스크립트 재 기입하지 않고 데이터베이스 수정 등 용도 항체, 부피, 및 대상으로 항체 분배 프로세스 상세의 번호를 변경할 수 있도록 융통성을 제공 하였다. 개념 실험의 증거 분석 세포 계측법 11 색, 17 항체 흐름의 복제 준비에 의해 입증 뛰어난 인터 및 인트라 분석 정밀도를 달성했다. 이 방법론은 분석 유동 세포 계측법에 대한 전체 처리량이 증가하고 상세한 pheno에 필요한 복잡한 항체 칵테일을 매일 준비를 촉진갓 수집 된 항 응고 말초 혈액의 typic 특성.

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

사람 말초 혈액의 Immunophenotypic 분석에 대한 항체 칵테일을 준비하는 반 자동화 된 접근

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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Research

JoVE Journal

Immunology and Infection

10.7K 조회수.  Providence Cancer Center, Providence Portland Medical Center. Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

비디오: A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

연구

면역학 및 감염병학

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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 ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

타액 항체 멀티 플렉스 면역을 개발하는 것은 환경 병원체의 인체 노출을 측정하는

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

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 ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...