보조금 지원이 작품은 과학 연구 (FNRS), 레 미스 드 난 연구소 보르, FNRS 조작 Télévie, 벨기에의 계획 암, 퐁 Lambeau-Marteaux, 퐁 JC Heuson과 퐁 Barsy에 대한 벨기에 기금 폴더 만.

참고 : 모든 표본은 각 환자에서 얻은 정보를 담은 서면 동의 연구소 쥘 보르 데의 의료 윤​​리위원회의 승인을 프로토콜을 사용하여 획득 하였다. 조직 파쇄 1. 준비 <ol><li> 절제된 조직 (수술실에서 절제 악성 및 정상 조직)를 해부 즉시 픽업 훈련받은 사람이 병리 실험실에 있습니다. 종양, NANT는 (가능한 한 종양에서 가장 먼 거리를 촬영) 및 정상 조직의 조각은 정기적으로 (1) 내에 처리 - 인체 조직에 대한 표준 바이오 안전성 절차를 사용하여 BSL2 실험실에서 수술 절제의 3 시간. 프로토콜의 흐름도는도 1에 도시되어있다. </li><li> 모든 조직편 (정상 및 종양 NANT)을 달아 길이, 폭, 높이 (길이 x 폭 x 높이)를 측정한다. 이는 세포의 개체군에 대한 후속 정규화 중요한 단계, 추출 된 RNA 등이며 NOTE : 샘플 크기의 범위는 더 지방이 가능하다면 100 10,000 mm의 3입니다. </li><li> 조직이 실제로 종양의 일부임을 확인하기 위해 H &amp; E 염색 용 유리 슬라이드에 종양 조각을 각인. <ol><li> 종양 조각에 유리 현미경 슬라이드를 누르면 몇 초 동안 손가락으로 부드럽게 압력을 적용하여이 작업을 수행합니다. </li><li> 물에 세척 과정에서 다음 2 분 동안 이소프로판올과 슬라이드를 수정합니다. 마이어의 헤 마톡 실린 30 초 동안 조직을 Counterstain과. </li><li> 물 여섯 화장실에서 슬라이드를 씻으십시오. 15 초 동안 Phloxine B 2 % 얼룩. </li><li> 물 한 욕조와 이소프로판올 및 마무리의 네 화장실 의해 물 한 욕조에 씻으십시오. </li><li> 1 분 드레인에 대한 이소프로판올에 품어. 자일 렌의 두 화장실에 걸린. </li><li> 자일 렌을 기반으로 장착 매체를 마운트합니다. 종양 세포 수 (그림 2)에 대한 검사. 참고 : - 기질, 림프 또는 광고 주로 종양 세포가 각인 된 슬라이드에 충실ipose 세포는 거의 종양 세포 사이에 공간을두고, (그림 2) 남아. </li></ol></li><li> 멸균 사용 - 화학적으로, 무 혈청 조혈 세포 배지 1 ㎖를 함유하는 작은 배양 접시에서 조직 단편을 배치 작은 조각 (2 ㎜ × 2 ~ 1)로 그것을 실온에서 (이하 매체 라 함)와 주사위 메스. </li><li> 기계 dissociator의 C 관 (조직 조각 + 매체) 모두를 전송합니다. </li><li> 파스퇴르 피펫을 사용하여 매체의 2 ㎖로 배양 접시와 메스를 씻어 C 관 (해리에 대한 매체의 볼륨 = 3 ㎖)이 추가. </li><li> 단일 세포 현탁액에 조직 조각을 균질화 C 튜브의 기계적 dissociator 프로그램 A.01 (가장 부드러운 프로그램)를 사용합니다. (한 사이클 = 25 초) 장치에서 C 튜브를 놓고 두 번 연속으로 프로그램을 실행합니다. 참고 :이 균질화 절차는 설립과 인간의 유방 조직, 다른 다치 대한 검증되었습니다또는 또는 조직 유형이 다른 프로그램을 사용해야 할 수도 있습니다 먼저 테스트해야합니다. </li><li> 장치에서 C 튜브를 제거하고 50 ML 튜브에 앉아 40 μm의 셀 스트레이너에 직접 균질을 가만히 따르다. 단계 1.6에서와 동일한 파스퇴르 피펫을 사용하여, 셀 스트레이너로 C 튜브에 남아있는 액체를 옮긴다. </li><li> 1 ㎖의 마이크로 피펫 팁을 사용하여 15 ML 튜브에 여과 된 액체를 전송합니다. 일시적으로 셀 스트레이너 및 50 ml의 튜브를 유지. </li><li> 배지 3 ㎖로 추가로 C 튜브를 헹구고 여전히 다시 50 ㎖ 튜브에 장착 셀 스트레이너로, 단계 1.6에서와 동일한 파스퇴르 피펫을 사용하여,이 전송. 부드럽게 연속적으로 용출액 오염을 막기 위해 폐기 된 깨끗한 파스퇴르 피펫 또는 1 ml의 팁을 사용하여 여과기 주위를 이동하여 50 ㎖ 튜브에 unhom​​ogenized 조직에 갇혀 잔류 액의 최대 양을 짜내. </li><li> 거꾸로 t의 셀 스트레이너를 배치그는 원래 C 튜브와 unhom​​ogenized 조직은 다시 C 튜브에 떨어질 수 있도록 매체의 3 ㎖ 씻어. </li><li> A.01 프로그램의 두 사이클 단계 1.7로 다시 균질화. </li><li> 50 ML 튜브에 앉아 셀 스트레이너를 통해이 두 번째 균질을 붓고, (단계 1.10에서와 같이) 3 ㎖ 매체를 다시 C 튜브를 씻어 다시 최대 압박 50 ML 튜브에 앉아 셀 스트레이너에 파스퇴르 피펫으로 전송 셀 스트레이너에 갇혀 잔류 결합 조직으로부터 액체의 양. </li><li> 이 시점에서, ~ 2.5 ml의 부피는 15ml의 튜브이고 ~ 50 ㎖ 튜브에서 9 ㎖의. </li></ol> 조직의 상층 액과 세포의 2. 분리 <ol><li> 실온에서 600 XG에서 15 분 동안 15 ㎖ 중의 균질 물 및 50 ㎖ 튜브를 원심 분리기. </li><li> 깨끗한 튜브에 15 ML 튜브에서 SN을 가만히 따르다 일시적으로 4 ℃에서 저장한다. 이 뜨는 = 차 종양, NANT 또는 정상 TIssue SN (최종 부피 2.5 ml)을 연속적으로 정화와 미래 분석 (아래 참조) -80 ° C에서 저장 이전에 분주합니다. </li><li> 50 ML 튜브에서 상층 액을 제거한다. </li><li> 부드럽게 1ml의 배지의 최종 부피 모두 세포 펠렛을 재현 탁. 요약하면, 첫째 부드럽게 (딱딱한 표면에 튜브를 눌러) 모두 튜브에 세포 펠렛을 휴식. 배지 500 μL와 50 ml의 느슨한 세포 펠렛을 재현 탁하고 제 펠렛을 재현 탁하는 15ml의 튜브에이 세포 현탁액을 전송. 세포의 최대 복구를위한 매체의 두 번째 500 μL 한 번이 단계를 반복합니다. </li><li> 혈구를 사용하여 생존 세포의 수를 계산하고 : 작은 튜브로 전송 세포 현탁액 10 μL은 트리 판 블루 10 μL (희석 한 1)과 혼합한다. 주 :이 시점에서 세포 현탁액의 분획은 또한 셀 크기, 입도를 평가하는 유동 세포 계측법에 의해 분석 될 수 있고 더 PREC 모집단에 대한 마커의 수가 제한 원한다면이전에 광범위한 분석이나 실험에 균질의 상대적인 세포 분포의 이세 평가. 흐름에 의해 모든 분석은 세포 계측법 개체군의 정상화 CD45 라벨을 통합합니다. </li><li> 실온에서 10 분 동안 300 XG에서 원심 분리하여 세포를 펠렛. 종양, NANT, 또는 정상 조직에서 셀은 추가의 정제 또는 분석을위한 준비가되어 있습니다. 수술과 같은 날에 수행 할 때 이러한 추가 단계가 최상이다. 주 : 유세포 분석뿐만 즉시 1 초 동안 볼 텍싱, 세포 펠렛을 적혈구 세포 용해 완충액 0.4㎖를 첨가하고 방에서 10 분의 최소 배양하여 항체 라벨링 후 용해되어야 잔류 적혈구 세포 분류되지 분석하기 전에 온도 (빛으로부터 보호). </li></ol> 조직 뜨는 3. 명확화 <ol><li> 4 ℃에서 15 분 동안 15,000 XG에서 조직 SN와 1.5 ml의 튜브를 원심 분리기. </li><li> 조심스럽게 t 제거그는 만지거나 펠렛을 방해하지 않고 뜨는. 수와 원하는 분량의 볼륨에 따라 깨끗한 튜브 (또는 튜브)에 전송합니다. </li><li> 향후 사용을 위해 -80 ° C에서 뜨는을 저장합니다. </li></ol> 4. 환자의 혈액 <ol><li> 헤파린 튜브에 정맥 천자에 의한 제어 수술 전에 일 각 환자의 혈액 샘플을 수집합니다. 혈장과 버피 코트를 얻기 위해 브레이크 오프 20 ºC에서 10 분 동안 400 XG에 혈액을 원심 분리기. </li><li> 혈장을 분리하고 20 ºC에서 15 분 동안 10,000 XG에서 원심 분리하여이를 명확히. 향후 사용을 위해 -80 ° C에서 나누어지는 및 저장 플라즈마. 매체에서 버피 코트를 희석 </li><li> 즉시 분석 모집단 분리, DNA / RNA / 단백질 추출 또는 동결에 앞서 표준을 Ficoll-hypaque 구배 원심 분리를 이용하여 단핵 세포를 분리. </li></ol> 5. 유동 세포 계측법 <ol><li> 제조에 따라 라벨 세포빛으로부터 보호 4 ° C에서 R의 지침. 세포 펠렛을 세포 용해 완충액 0.4 mL를 넣은 후 표지 항체 조직편으로부터 세포 현탁액를 Lyse 적혈구. </li><li> 소용돌이는 즉시 1 초간 및 빛으로부터 보호 실온에서 10 분의 최소, 부화. 세척하지 않고 데이터 수집을위한 유동 세포 계수기에 표시된 세포를 전달합니다. </li></ol>

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The authors declare that no conflict of interest exists.

이 연구는 이후의 세포 분류, 추출, 동결 보존 및 / 또는 CD45 + 개체군의 표현형 분석을위한 소화 효소없이 정상 및 악성 유방 조직 균질의 빠른 준비를위한 최적화 된 방법을 설명합니다. 이 실험적인 접근의 목표는 밀접하게 자신의 생체 상태를 반영하고 수술실에서 신선한 조직의 최소한의 조작으로 정상 조직에 비교 TIL의 이미지를 생성하는 것입니다. 지금까지, 우리의 실험실&gt...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:409px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs Dissociator&#160;</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-093-235</td>
			<td>BD Medimachine is somewhat equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5810 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Centrifuge 5417 R</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard microcentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Esco Class II A2 Biosafety Cabinet</td>
			<td>ESCO global</td>
			<td style="width:137px;">N/A</td>
			<td>or other standard BSL2 hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted Microscope</td>
			<td style="width:163px;">Nikon eclipse TS100</td>
			<td style="width:137px;">N/A</td>
			<td>or other microscope compatible for a hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">B&#252;rker Chamber</td>
			<td style="width:163px;">Marienfield&#160;</td>
			<td style="width:137px;">640210</td>
			<td>or other standard hemacytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Navios Flow Cytometer</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">N/A</td>
			<td>or other flow cytometer (8-10 color recommended)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Materials</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">GentleMacs C-Tube</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-344</td>
			<td>BD Medimachine uses Filcon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell Culture Dish</td>
			<td style="width:163px;">Sarstedt</td>
			<td style="width:137px;">72,710</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Disposable Scalpel</td>
			<td style="width:163px;">Swann-Morton</td>
			<td style="width:137px;">0510</td>
			<td>or standard single use sterile scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Cell Strainer 40&#181;m</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">734-0002</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 50mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352070</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD Falcon Tube 15mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352097</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BD FACS Tube 5mL</td>
			<td style="width:163px;">Becton Dickinson</td>
			<td style="width:137px;">352008</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sterile Pasteur Pipette 5 mL&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">612-1685</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Microfuge Tube 1.5 mL</td>
			<td style="width:163px;">Eppendorf</td>
			<td style="width:137px;">7805-00</td>
			<td>or other non-pyrogenic plasticware&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;"></td>
			<td style="width:163px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:272px;">Reagents</td>
			<td style="width:163px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">X-Vivo 20</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE04-448Q</td>
			<td>serum-free medium recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Phosphate buffered saline</td>
			<td style="width:163px;">Lonza</td>
			<td style="width:137px;">BE17-516F</td>
			<td>standard physiological PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Trypan blue&#160;</td>
			<td style="width:163px;">VWR</td>
			<td style="width:137px;">17942E</td>
			<td>or other vital stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">VersaLyse</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">A09777</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fixable viability Dye eFluor 780&#160;</td>
			<td style="width:163px;">eBioscience</td>
			<td style="width:137px;">65-0865-14</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 FITC</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345763</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD3 Vio Blue</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-094-363</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 PE</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345769</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD4 APC</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-091-232</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 ECD</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:137px;">737659</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD8 PerCP</td>
			<td style="width:163px;">BD Biosciences</td>
			<td style="width:137px;">345774</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD19 APC-Vio770</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-643</td>
			<td>for flow cytometry experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-CD45 VioGreen</td>
			<td style="width:163px;">Miltenyi Biotec</td>
			<td style="width:137px;">130-096-906</td>
			<td>for flow cytometry experiments</td>
		</tr>
	</tbody>
</table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

선천성 및 후천성 면역 반응 모두에서 종양 탈출 특징으로 면역 시스템을 무력화하는 악성 세포의 능력은 지금 암의 중요한 특징으로 받아 들여진다. 유방암에 대한 우리의 연구는 종양 침윤 림프구가 종양의 진행과 환자 결과에서 재생하는 적극적인 역할에 초점을 맞추고있다. 이 목표를 향해, ​​우리는 그들의 원래 상태로 그들을 근접 평가하기위한 노력의 일환으로 정상 및 비정상 조직에서 그대로 림프 세포의 신속한 분리를위한 방법론을 개발했다. 표면 수용체 발현 비교하는 효소 - 소화 된 조직을 유지하면서 기계적 dissociator 쇼 모두 증가 된 생존율 및 세포를 회수하여 제조 균질. 또한, 나머지 불용성 물질의 소화 효소는 기본 균질의 양적, 질적 측정 가능성이 진정으로 조직 fragm에 침투 부분 집단을 반영하는 것을 나타내는 추가 CD45 + 세포를 복구하지 않았다천만에. 이러한 균질 림프 세포는 쉽게 면역 (표현형, 확산 등) 또는 분자 (DNA, RNA 및 / 또는 단백질)을 이용하여 특성화 할 수 접근한다. CD45 + 세포는 모집단 정화 시험 관내 팽창 또는 냉동 보관을 위해 사용할 수있다. 이 접근법의 또 다른 장점은 균질 물에서 뜨는 기본 조직과 정상 조직에서 악성 사이토 카인, 케모카인, 면역 글로불린 및 본 항원을 특성화하고 비교하기 위해 사용될 수 있다는 점이다. 이 프로토콜 기능을 매우 잘 인간 유방 조직 및 정상 및 비정상 다양한 조직에 적용 할 수 있어야한다.

시판 티슈 해리 용액 또는 콜라게나 제의 다양한 실험 혼합물의 DNase 및 / 또는 히알루로니다 제 억제제 중 하나와 조직 절편의 소화 효소는 세포 표면 수용체의 다양한 절단. 최초 유방 종양 침윤 CD4 + T 세포에 대한 우리의 연구가 집중, 신속 인해 소화 효소 표준 프로토콜을 사용하여 4-8 표면 CD4 수용체의 절단에 중요한 기술적 문제를 제시 하였다. 우리는 높은 생존 성 림...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human

Research

JoVE Journal

Immunology and Infection

19.9K 조회수.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

비디오: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

에서 DNA 추출을위한 간단한 Chelex 프로토콜<em&gt; 아노펠레스</em&gt; 종.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

의 혈소판 응집 중재를위한 간단한 프로토콜<em&gt; 변형체 falciparum의</em자원별로 설정에서&gt; - 감염된 적혈구

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

인간 팔라티노 편도선에서 B와 T 림프구에 대한 효율적인 분리 프로토콜

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated 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 ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

혈액 처리를위한 빠른 방법은 인간의 혈액에서 혈장 펩타이드 수준의 수율을 증가하는

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

간단하고 효율적인 접근 방법은 돌연변이 백시 니아 바이러스 벡터를 구축하기

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

최적화 된 프로토콜은 림프구에 해당 작용과 미토콘드리아 호흡을 분석하는

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

면역 마약 식별을위한 T 림프구에서 미토 겐 유도 블라스의 신속한 정량

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...