This work was made possible by funding from the German Federal Ministry of Education and Research (BMBF, 1315883) as well as by the S&#228;chsische Aufbaubank (SAB) project 100116526 from a funding of the European Regional Development Fund (ERDF).

모든 인간의 혈액 샘플을 건강한 지원자로부터 획득하고,인가 된 백혈구 농축 프로토콜은 라이프 치히 대학의 의학 교수의 윤리위원회의 지침을 따릅니다. 쥐의 혈액과 실험 동물 관리 및 사용에 독일의 지침에 따라, 책임 지역 윤리위원회 (Landesdirektion 작센, Referat 24)에 의해 승인되었다. 1. 실험 설정 주 : 혈액 샘플에서 적혈구의 고갈에 대한 저장성 용해 절차는 시간이 중요한 과정이기 때문에, 사전에 프로토콜의이 부분에 필요한 모든 장치 (예를 들면, 완충액)을 제조 하였다. <ol><li> 저장성 용해 하나 15 ml의 원심 분리기 튜브와 혈액 샘플 당 후속 아미노 페닐 플루 오레 (APF) 염색 한 1.5 ml의 샘플 튜브 라벨. <ol><li> 백혈구 보충은 멸균 조건 하에서 수행 할 경우, 흐름 하에서 라벨링을 수행상자. </li></ol></li><li> 샘플 당 약 12​​ ml의 인산염 완충 식염수 (PBS, 10 mm)를 준비합니다. 백혈구는 멸균 조건 하에서 격리 여부되어야하는지 여부에 따라 공급의 준비 멸균 용액을 사용하거나 증류수 PBS 정제를 용해하거나 PBS를 준비한다. pH 값을 확인하고, 필요한 경우, 0.1 M 염산 및 수산화 나트륨 용액의 소량을 사용하여 pH를 7.4로 조정한다. <ol><li> 약 16 샘플에 대한 충분한 비 멸균 완충 용액을 얻었다 증류수 200 ㎖ 중의 (자재 표 참조) PBS 정제 녹인다. 원하는 경우, 멸균 여과하여 용액을 소독. </li></ol></li><li> 칼슘 보충 약 1 ml의 행크스 균형 염 용액 (HBSS) 2 + 샘플 당을 준비합니다. <ol><li> 100 ㎖의 HBSS 소금 (최종 부피)를 두 번 증류수 970 mg을 녹이고. 최종 부피까지 충전하기 전에, pH 값을 확인하여 pH 7.4로 조정한다. 낮은 버퍼 용량으로 인해, 매일 갓이 버퍼를 준비0.1 M 염산 및 수산화 나트륨 용액을 소량 사용하여 특별한주의와 pH 조정을 수행 할 거라고. 참고 멸균 용액은 실험에 따라 사용될 수있다. 용액을 멸균 여과에 의해 멸균 될 수있다. </li></ol></li><li> 두 버퍼 라벨 게다가와 튜브를 준비 (예를 들어, 50 ㎖ 원심 분리 관) 또는 플라스크 (예를 들어, 측정 컵 250 ㎖)에 미리 용해 저장성 절차 이중 증류수. 샘플 당 약 10 ㎖의 물을 준비합니다. </li><li> 얼음에 수행되는 APF 염색 (섹션 3), 분쇄 얼음 용기를 준비합니다. </li><li> 염색시 사용 작업 솔루션을 신속하게 준비 할 수 있도록 사전에 APF의 분취 량을 준비 (3.2 단계) 및 냉동와 APF 솔루션의 해동을 반복하지 않도록. 주 : APF는 일반적으로 5 ㎎ / ㎖의 메틸 아세테이트 (11.81 mM)을 원액으로 얻어진다. <ol><li> 0.5 ml의 튜브에 10 ~ 100 μL의 분취 량을 동결. APF는 최종 염색을 위해10 μM의 염료 농도 (단계 3.8)를 사용한다. </li></ol></li></ol> 적혈구 2. 저압 성 용해 주 : 오염을 피하기 위해 층류 벤치에서 (원심 단계 제외) 저장성 용해 공정을 수행한다. 실온에서 전체 절차를 수행한다. 근력 저하 용해 시간이 중요한 공정이기 때문에, 사전에 물 (2 mL) 및 PBS (5 ㎖)에 첨가 피펫을 조정 절차를 시작하기 전에 물과 PBS 플라스크 연다. 사용하기 전에 실온에서 PBS 용액과 증류수 저장한다. <ol><li> 각각의 혈액 샘플 전송에서 해당 15 ML의 원심 분리 관에 100 μL. 참고 : 본 실험에서 혈액 시료는 일반적으로 응집을 방지하기 위하여 헤파린 10 U / ㎖를 함유한다. 그러나 의한 샘플 물질의 다른 소스에 항응고제의 특성 및 농도는 다를 수있다. APF 염색에 미치는 영향은 비교 예에 의해 테스트해야다른 유형 또는 항 응집제의 농도로 보충 된 혈액 샘플로부터 얻은 결과와 그 결과를 보내고. </li><li> 2 ml의 증류수를 추가하고, 중간 레벨로 설정 볼텍스 혼합기를 사용하여 샘​​플을 혼합한다. 다음 샘플 당 5 ml의 PBS를 추가하고 와류 믹서를 사용하여 혼합, 60 초 동안 샘플을 품어. 주의 : 샘플을 약 5까지 병렬로 제조 될 수있다. 샘플 순서 비교 배양 시간을 달성하기 위해 물 및 PBS를 첨가 동일하게 유지. </li><li> PBS를 첨가한다 (단계 2.2)을 실온에서 6 분, 450 × g으로 원심 분리하여 모든 샘플에서 남은 그대로 펠렛 세포에 의한 등장 성 상태의 재생 후. </li><li> 테이블 폐기물 빈으로 쏟아져하여 상층 액을 제거합니다. 원심 분리 튜브를 반전하고 종이 타월 위에 개방을 눌러 많은 액체를 가능한 한 제거합니다. 세포 펠렛은 가능한 한 건조되었는지 확인합니다. </li><li> (단계 2.2) 이전에 설명 된대로 저장성 용해 절차를 반복합니다. 엔OTE는 원칙적으로 본 저장성 용해 절차 얻어지는 혼합 백혈구 분획의 순도에 따라 두 번 이상 반복 될 수있다 (2.5-2.3이 단계). 두 용해가 혼합 얻어진 세포 분획에서 적혈구 잔존 주 단계 후에 약 25 %이다. </li><li> 실온에서 6 분, 450 × g으로 원심 분리에 의해 나머지 본래 세포 펠렛. 뜨는 (단계 2.4 참조)를 제거합니다. 펠릿에 500 ㎕의 HBSS를 추가하고 균일 한 용액이 얻어 질 때까지 부드럽게 녹인다. 따라 표시 1.5 ml의 샘플 튜브에 각 샘플을 전송합니다. </li><li> 실험에 따라서 직접 (유동 세포 계측법을 통해 예) 얻어진 샘플 25 (32) (단일 세포 유형의 식별을위한) 형광 표지 된 항체로 염색 분석 (세포 활성화 실험) 세포 자극으로 33 부화 또는 얼음 사용에 저장된 후에. 연구의 목적에 따라 즉시 후속 실험을 수행과립구 보낸 혼합 백혈구 분획은 약 20 시간의 매우 짧은 반감기를 갖는다. 참고 :이 또한 아래에 설명 된 퍼 옥시 다제 활성 염색을 위해 보유하고있다. </li></ol> 3. 할로겐 퍼 옥시 다제 활동 염색 참고 : 혈액 유래 헴 퍼 옥시다아제 MPO 및 EPO에 의해 HOCl- 및 HOBr 생산 명명 된 hypohalous 산에 의해 형광에 산화 APF를 사용하여 정량한다. 형광 표지 된 항체와 세포 라벨링 APF 염색과 조합하여 수행되는 경우에 따라서, 형광의 발광 신호에 간섭을 피하기 형광체. <ol><li> 얼음에 4 ° C에서 APF 염색을 수행합니다. </li><li> APF의 작동 용액을 제조하는 염료의 적절한 분액을 해동 (예, 10 μL, 11.81 mM)을하고 정확하게 일 밀리미터 (예를 들면, 10 μL에 108.1 μl의 완충액을 추가)하는 HBSS로 희석. <ol><li> 각 샘플 (500 ㎕의 세포 용액) 5 μ 준비설명 APF 작업 용액의 리​​터. 따라서, 118.1 μL APF 작업 용액 (1 ㎜)이 약 20 샘플을 준비하는 산출 APF (11.81 mM)을 하나 10 μL 나누어지는을 사용합니다. 때문에 피펫 동안 손실을 계산보다 약 10 % 더 APF 작업 솔루션을 준비합니다. </li></ol></li><li> 상기 APF 염색의 과산화 효소 특이성을 확인 헴 퍼 옥시 다제 억제제 4- 아미노 벤조산 히드라 지드 (4- ABAH) 35 제어 샘플을 준비하기 위해. <ol><li> 용매 500 μL에 756 mg의 4- ABAH 희석하여 디메틸 술폭 시드 4- ABAH (151.17 g / 몰) (DMSO)에 1 M 스톡 용액을 제조 하였다. 또한 HBSS에서 4 ABAH 1/10 희석. </li></ol></li><li> 각각 &quot;70 mM의 H 2 O (2)&quot;과 &quot;7 mM의 H 2 O (2)&quot;을 가진 H 2 O이 작동 용액 라벨 개의 1.5 mL의 원심 분리 튜브의 제조. <ol><li> &quot;70 mM의 H 2 O 2&quot;로 표시된 튜브에서 갓 10 μl를 희석약 88 mM의 농도를 얻기 위해 증류수 990 μL를 사용하여 H 2 O (2)의 30 % 스톡 용액 (8.8 mM)을 중. 얼음에 보관하고 4 시간 이내에 사용한다. 참고 : H 2 O 2는 일반적으로 공급 업체에 의해 30 % 원액으로 전달된다. 4 ℃에서 저장 될 때, 그것은 약 3 년 동안 안정하다. 분해를 방지하기 위해 증류수에 H 2 O 2의 희석을 수행합니다. (2) H 2 O 희석은 0.1 M NaOH 용액에서 제조 될 수있다. </li><li> 정확한 H를 결정하기위한 2 O 2 농도로 레이블 된 1.5 ㎖의 튜브에 증류수 900 ㎕를이 용액 100 μl를 첨가함으로써 처음으로 H 2 O 2 스톡 용액 (약 88 mm)를 추가로 1 ~ 10의 희석을 준비 &quot;7mm의 H 2 O 2&quot;. </li><li> (AN-UV 비스 photospectrometer를 사용하여 근거리 UV 범위 (예를 들면, 200 ~ 300 ㎚)에서의 스펙트럼을 기록하고 240 nm에서 흡광도를 사용49 240 = 34 M -1 cm-1)가 제 2 H 2 O 2 원액 (34)에 실제 H 2 O 2 농도를 결정한다. 기준 큐벳은 증류수가 들어 있는지 확인합니다. 측정에 사용하는 석영 큐벳을 위해 UV 영역에서 스펙트럼을 기록되어있다. </li><li> 이 결과로부터 모두 H 2 O 2 스톡 용액의 정확한 농도를 계산한다. 증류수 적당량 첨가함으로써 &quot;70mm의 H 2 O 2&quot;샘플 튜브 정확히 70mm의 상기 제 원액의 농도를 조정한다. 얼음에 얻어진 작업 솔루션을 저장하고 1 시간 이내에 사용한다. </li></ol></li><li> 1 mm의 최종 농도에 해당하는 샘플 4 ABAH 작업 용액 (100 mM)을 5 μl를 추가합니다. APF 추가하기 전에 배양기에서 37 ° C에서 15 분 동안 샘플을 품어. </li><li> 각 5 μL APF 작업 용액 (1 ㎜)를 추가500 ㎕의 샘플을 10 μM의 최종 염료 농도가 얻었다. (중간 설정에서 볼텍스 믹서를 사용하여, 예) 샘플을 부드럽게 혼합하고 37 ° C에서 30 분 동안 배양한다. </li><li> 700 μM의 최종 농도 각 500 μL 시료에 5 μL의 H 2 O 2 작동 용액 (70 mm)를 추가합니다. 부드럽게 샘플을 혼합하고 37 ℃에서 60 분 동안 그들을 품어. 병렬로 적절한 제어 측정을 수행한다. 주 : 제어 측정은 700 μM H 2 O (2) 세포에 대한 독성이없는 것으로 나타났다. 도 현저한 세포 반응이 관찰되었다. 주 : APF 염색 또한, H 2 O 2의 첨가를 생략 과학적 문제에 의존하여 수행 될 수있다. H (2) O (2)의 첨가는 세포에 의한 최대의 HOCl 및 HOBr 생산의 검출을 허용하면서 과산화수소가없는 경우에만 기저 염소 EPO 및 MPO 활성이 결정된다. 티그는 후자 상당히 높은 형광 신호를 의미한다. </li><li> 세포 펠렛 들어 10 실온에서 분, 400 X g 위해 원심 분리. 철저하게 펠렛을 파괴하지 않고 뜨는을 제거하고 세포를 재현 탁 250 ㎕를 HBSS를 추가합니다. 참고 : 샘플은 현재 25 유동 세포 계측법을 통해 분석을위한 준비가되어 있습니다. </li><li> 표백을 피하기 위해 분석 될 때까지 어둠 속에서 샘플을 저장합니다. 480-490 nm에서 여기 후 약 525 nm의 37에서 형광의 방출을 감지합니다. </li></ol>

The authors have nothing to disclose.

호중구를 인간 혈액에서 가장 풍부한 백혈구과 마찬가지로 퍼 옥시 다제 양성 세포의 분리는 종종 이러한 셀에 초점을 밀도 구배 원심 분리 (38)에 의해 다른 백혈구에서 호중구의 분리를 포함한다. 호중구는 훨씬 덜 풍부 뮤린 혈액 샘플에 대해서는 아직 후자보다 복잡한 방법 39 40을 사용한다. 또한 두 방법 모두는 또한 더 큰 혈액 볼륨의 필요성, 샘플에서 퍼 옥시 다제 ?...

다형 핵 백혈구 (백혈구라고도 과립구) 및 단핵구는 혈액 -1,2-의 선천 면역 세포의 중요한 구성 요소를 나타낸다. 이들은 병원균뿐만 아니라, 획득 면역 시스템의 활성화 및 전신성 염증 반응 2-4 개시까지 기본 방위에 기여한다. 그러나, 특히 호중구 과립구의 가장 풍부한 형태 및 단핵구는 크게 급성 염증성 사건 (5)의 규제와 종단에 기여한다. 따라서,이 세포는 류마티스 관절염 -6,7- 같은 만성 염증성 질환에서 중요한 역할을 할 수있다. 사실, 천식, 만성 염증성기도 질환, 호산구의 세포 사멸을 손상 혈액 여덟 번째 대부분 과립구 형 특징이다. 그러나 과립구의 세포 사멸 및 대 식세포에 의해 자신의 빠른 제거는 휴대​​ 종료하는 동안 두 가지 중요한 단계는염증 9-11의. 명명 된 면역 세포 두 밀접하게 관련 효소, 즉 마이 엘 로퍼 옥시 다제 (MPO, 호중구와 단핵구)와 호산구 퍼 옥시 다제 (EPO, 호산구)에서 12, 13에서 찾을 수 있습니다. 이 헴 퍼 옥시다아제는 고전 그들이 두 전자적으로 해당 저자 (의사) 내지 (의사 -) 할로겐 자신의 살균 특성 14-16 알려져있다 halous 산을 산화로 체액 성 면역 반응 관련이 있습니다. 생리 학적 조건 MPO 주로 양식 차아 염소산 (의 HOCl)과 hypothiocyanite (- OSCN) 후자와 하이포 아 브롬 산 (HOBr)이 EPO 17-19로 형성되어있다. 새로운 결과는이 (의사 -) 할로겐 효소 활성은 또한 염증 반응의 조절과 면역 반응 (20, 21)의 종료에 기여할 수 있음을 시사한다. 사실, MPO 및 가공 제품의 HOCl 의해 생산 T 세포 기반 적응 면역 반응을 억제 22-2 나타났다4. 이 생리적 기능 MPO와 EPO의 기여를 만성 염증성 질환의 선천 면역에서 백혈구의 면역 학적 역할에 대한 추가 통찰력을 얻을 수를 결정하기 위해 우리는 빨리 후속 특정 소형 혈액으로부터 백혈구를 농축하는 방법을 개발 이들 세포의 할로겐화 퍼 옥시 다제 활성의 결정. 적혈구 고갈 위해 우리는 낮은 재료 비용으로 빠른 백혈구 농축에 이르게 증류수, 두-이후 저장성 용해 단계를 포함하는 표준화 된 방법을 선택했습니다. 후속 할로겐 MPO의 결정 및 EPO의 활동에 대한 HOCl- 및 HOBr 특정 염료 아미노 페닐 플루 오레 (APF)는 25 ~ 27을 사용 하였다. 퍼 옥시다아제 비특이적 염색 방법 28,29 적용 달리,이 방법은 종종 심각한 손상 infl에 할로겐 옥시다아제 활성의 선택적 검출을 허용ammation 30, 31.

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">materials/equipment</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">15 ml centrifugation tubes</td>
			<td style="width:203px;">VWR/Corning</td>
			<td style="width:120px;">734-0451</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">1.5 ml sample tubes</td>
			<td style="width:203px;">VWR/Eppendorf</td>
			<td style="width:120px;">211-2130DE</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:213px;">Pipettes for volumes up to 5 ml</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">e.g. 3120000070</td>
			<td style="width:203px;">We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 &#181;l, 10-100&#181;l, 100-1000 &#181;l an 500-5000&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">laminar flow bench</td>
			<td style="width:203px;">Thermo Electron Corperation</td>
			<td style="width:120px;">HeraSafe</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Vortex mixer</td>
			<td style="width:203px;">Bender &#38; Hobein AG</td>
			<td style="width:120px;">Vortex Genie 2</td>
			<td style="width:203px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tabletop centrifuge</td>
			<td style="width:203px;">Kendro Laboratory Products</td>
			<td style="width:120px;">Laborfuge 400R</td>
			<td style="width:203px;">The centrifuge should be able to be used at 450x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Small centrifuge</td>
			<td style="width:203px;">eppendorf</td>
			<td style="width:120px;">5415D</td>
			<td style="width:203px;">The centrifuge should be able to be used at 400x g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Incubator</td>
			<td style="width:203px;">Heraeus</td>
			<td style="width:120px;">cytoperm 2</td>
			<td style="width:203px;">Settings: 37 &#176;C, 95% humidity, 5 % CO2 content</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">UV-Vis spectrophotometer</td>
			<td style="width:203px;">Varian</td>
			<td style="width:120px;">Cary 50 bio</td>
			<td style="width:203px;">A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Flow cytometer</td>
			<td style="width:203px;">Becton, Dickinson</td>
			<td style="width:120px;">BD Facs Calibur</td>
			<td style="width:203px;">Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g 488 nm argon laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Chemicals</td>
			<td style="width:203px;"></td>
			<td style="width:120px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate buffered saline (PBS)</td>
			<td style="width:203px;">amresco</td>
			<td style="width:120px;">K812</td>
			<td style="width:203px;">sterile solution, ready to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;"></td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">P4417</td>
			<td style="width:203px;">tablets for solving in 200 ml millipore water</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Hanks balanced salt solution (HBSS) with Ca(2+)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1387</td>
			<td style="width:203px;">970 mg/100 ml, carefully check and adjust the pH value to 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:203px;">Merck Millipore</td>
			<td style="width:120px;">1.09057.1000</td>
			<td style="width:203px;">1M solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Sodium hydroxide</td>
			<td style="width:203px;">Riedel-deHa&#235;n</td>
			<td style="width:120px;">30620</td>
			<td style="width:203px;">Solid pellets. For a 1M solution solve 4 g/100 ml Millipore water</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Aminophenyl fluorescein</td>
			<td style="width:203px;">Cayman</td>
			<td style="width:120px;">10157</td>
			<td style="width:203px;">5 mg/ml solution (11.81 mM) in methyl acetate, aliquotes of e.g. 100 &#181;l should be prepared and stored at -20 &#176;C</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:213px;">Hydrogen peroxide</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">H1009</td>
			<td style="width:203px;">This 30% stock solution corresponds to a concentration of about 8.8 M. Further dilutions have to be freshly prepared in distilled water immediately prior to use and quantified by absorbance measurements</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">4-aminobenzoic acid hydrazide (4-ABAH)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">A41909</td>
			<td style="width:203px;">A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DMSO</td>
			<td style="width:203px;">VWR chemicals</td>
			<td style="width:120px;">23500.26</td>
			<td style="width:203px;">-</td>
		</tr>
	</tbody>
</table>

fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples

이 논문에서는 작은 전체 혈액 샘플에서 백혈구의 신속하고 표준화 된 농축을위한 프로토콜을 설명한다. 이 절차는 적혈구의 저 삼투압의 용해를 기반으로 비 - 인간 기원의 혈액뿐만 아니라 인간의 샘플에 적용될 수있다. 약 50 ~ 100 μL의 작은 초기 샘플 볼륨이 작은 실험 동물에서 반복 채혈에이 방법을 적용 할 수 있습니다. 또한, 백혈구 농축 여러 실험실 환경에서이 방법을 적용하고, 분 이내에 화학 물질 및 계측에 대한 낮은 재료 노력으로 얻을 수있다. 백혈구의 표준화 정제는 매우 선택적 헴 퍼 옥시다아제의 할로겐화 퍼 옥시 다제 활성을 평가하기 염색법, 마이 엘 로퍼 옥시 다제 (MPO)와 호산구 퍼 옥시 다제 (EPO), 즉, 차아 및 하이포 아 브롬 산 (의 HOCl 및 HOBr)의 형성과 결합된다. MPO 강하게 neut 표현되지만rophils 인간 혈액과 단핵 세포에 가장 풍부한 면역 세포 유형은 관련 효소 EPO 독점적 호산구 표현된다. 이러한 효소의 활성은 할로겐화 거의 HOCl- 및 HOBr 특정 염료 아미노 페닐 플루 오레 (APF) 및 차 퍼 옥시 다제 기질, 과산화수소를 사용하여 해결된다. 이후의 유세포 분석되면 모든 퍼 옥시 다제 양성 세포 (호중구, 단핵구, 호산구)를 구별하고 그들의 할로겐화 퍼 옥시다아제 활성을 정량화 할 수있다. APF 염색은 세포 표면 마커의 적용과 결합 될 수 있기 때문에,이 프로토콜은 특히 백​​혈구 하위 분획을 해결하기 위해 확장 될 수있다. 상기 방법은 인간 및 쥐 백혈구에 양의 HOCl 및 HOBr 생산 검출에 적용 할 수있다. 만성 염증성 질환에서 이러한 효소 제품의 광범위하고 다양하게 설명 면역 역할을 감안할 때, 이러한 프로토콜의 이해에 기여백혈구 파생 된 헴의 퍼 옥시다아제의 면역 학적 관련성.

이전에보고 된 바와 같이, 상술 한 방법은 인간 및 인간 이외의 물질 (32)에 모두 적용 가능한 것으로 밝혀졌다. 천식 증상이있는 쥐에 대해 표시 또한 같이 APF 염색은 전신 염증성 상태의 차이를 감지 할 수있는 적절한 도구가 될 수 있습니다. 따라서 우리는이 프로토콜을 사용하는 후속 연구에서 반복적으로 프리 스탄 유도 관절염 (PIA) 여성 다크 아구 티 쥐에?...

Watch this Scientific Journal Video about Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples at JoVE.com

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

백혈구가 풍부한 혈액 샘플의 할로겐 퍼 옥시 다제 활동의 신속하고 구체적인 평가

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

fast-specific-assessment-halogenating-peroxidase-activity-leukocyte

Research

JoVE Journal

Immunology and Infection

10.2K 조회수.  University of Leipzig. This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

비디오: Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

임상 항체 샘플 Phagocytic 활동 결정

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Monocytes과 인간 혈액의 작은 샘플에서 돌기 세포의 리니지 특정 수신자 같은 수용체 - 매개 시그널링의 정량 이미징

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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 ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...

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 ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

의 검출을위한 자석 교반기 방법<em&gt; 선모충</em근육 샘플에서&gt; 애벌레

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...