이 작품은 NIH 부여 P01 AI 068,730 (SNC, NS), NIH 부여 CA016520 / TAPITMAT (NS), Claneil 재단 (NS)에서 민간 자금 및 FCCC 및 펜실베니아 대학에 난소 포자 부여 (지원했습니다 P50 CA83638)와 폭스 체이스 암 센터 코어 그랜트 (P30 CA06927) (DCC). 저자는 펜실베니아 암 대학에서 박사 G. Danet - 데스노여스 감독 줄기 세포 및 이종 이식 코어에서 펜실베니아, 안토니 Secreto 대학에서 박사 EJ Delikatny 감독 광 / Bioluminescence 핵심 시설의 우수 기술 지원을 감사합니다 수술에 도움을 위해 펜실베니아 / OCRC 대학에서 orthotopic 분사 기술과 Denada Dangaj에 훈련 SNC 센터.

1. 세포 배양 <ol><li> orthotopic 사출, 문화 T175 플라스크에 DR26 종양에서 파생된 MOV1 캣 세포는, 그들의 90 %까지 합류하기 전에. 1 또는 2 T175 flasks 필요합니다 주입 당 1-5000000 세포를 이용할 계획입니다. </li><li> 주사 당일, 세포를 수확하고 hemocytometer를 사용하여 휴대폰 번호를 확인합니다. </li><li> 일단 세포 농도는 실온에서 300 X g에서 5 분간 원심 분리하여 세포 펠릿, 결정되었습니다. </li><li> 스핀에 따라, 0.002 M EDTA (에틸렌 다이아 민 테트라 초산)과 멸균 PBS 10 microliters 1 만달러에 전지를 resuspend </li></ol> 2. 사전 외과 <ol><li> 이전 수술, PBS EDTA (에틸렌 다이아 민 테트라 초산) 10 microliters에서 1,000,000 MOV1 - 캣 전지와 3/10cc 인슐린 주사기를 입력합니다. </li><li> 가열 패드에 isofluorane anesthetized 마우스를 전송합니다. 눈 탈수를 방지하기 위해 눈 연고를 추가합니다. 그런 다음, 즉시 수술 내내 마취를 제공하기 위해 isoflurane 기화기에 연결된 코 콘 시스템에 동물의 머리를 삽입합니다. </li><li> 알코올 면봉으로 주입 사이트의 소독 후, subcutaneously 3/10cc 인슐린 주사기와 함께, 5 MG / 케토프로펜, 사전 수술 진통제의 kg을 주입. </li><li> 가위를 사용하여 동물의 꼬리의 기본에 thoracolumbar 교차점에서 dorsum의 왼쪽 꼬리 부분을 면도. 완전히 머리를 제거하는 제모 크림을 적용합니다. 그런 다음, 젖은 종이 타월로 여분을 제거합니다. </li><li> 머리가 삭제되면, povidone - 요오드와 알코올 면봉으로 면도 영역을 소독. 그런 다음, 절개의 주변 외과 드레이프를 놓으십시오. </li></ol> 3. 외과 <ol><li> 그냥 수술 전에 1.5 %에 isoflurane 기화기 수준을 조정합니다. </li><li> 동물이 발을 패드 곤란하여 완전히 anesthetized되었는지 확인합니다. </li><li> 다음, 피부에서 비장을 찾습니다. 다음 외과 가위를 사용하면, 비장의 오른쪽 상단에있는 1~2센티미터 길이 dorsolateral 절개합니다. </li><li> retroperitoneum을 해부하다. 마우스 난소를 둘러싼 패드가 관찰됩니다. 마우스 난소를 둘러싼 지방 패드를 파악하고 노출 곡선 집게를 사용하십시오. </li><li> 멸균 PBS의 일부 방울과 수화물 기관. </li><li> 주사에 대한 난소를 파악 철회 ... 위치를 ... 그리고 안전하게 곡선 포셉를 사용하여 </li><li> 단단히 포셉과 난소를 쥐고 있지만, 난소에 MOV1 캣 종양 세포의 10 microliters을 주입. 회사 범위는 유체 병리 현상이나 누설을 방지할 수 있습니다. </li><li> 즉시 주사 다음, 포셉에 의해 끼쳤다 긴장을 풀어주고있다. 난소의 천공은 저절로 취소 및 종료해야합니다. </li><li> 바늘에 연결된 흡수성 Polyglycolic의 산성 봉합사를 사용하여 복고풍 - 복막 상처를 닫습니다. </li><li> 코 콘에서 동물을 놓습니다. </li><li> 피부를 스트레칭하고 조직 접착제의 몇 방울로 dorsolateral 상처 가장자리를 밀봉. </li><li> 마지막으로, 구두로 동물에 항생제 100 microliters을 관리할 수 있습니다. 그런 다음 복구에 대한 케이지와 모니터에 다시 놓으십시오. 일주일 동안 식수의 항생제에 쓰레기야. </li></ol> 4. 생체내 이미징에서 <ol><li> 일주일 MOV1 캣 종양 세포의 orthotopic 주사 후 생체내 이미징에서 수행합니다. 이미지 실로 isofluorane anesthetized 마우스를 전송하여 시작합니다. </li><li> 아래 2 % isoflurane 기화기 수준을 돌립니다. </li><li> 이미징 시스템 제조 업체의 지침에 따라 생체내 이미징에서 수행합니다. 이 비디오에서 루미나 시스템이 사용됩니다. </li><li> 이미지, 생활 이미지 소프트웨어 데스크톱 아이콘을 클릭하십시오. 그런 다음 IVIS 수집 제어판에서 &quot;초기화&quot;를 선택하십시오. 악기 설정은 카메라 설정에 유사한 아르 </li><li> 인수 제어판에있는 악기의 인수 매개 변수를 설정합니다. 형광 들어, &quot;형광&quot;을 선택하십시오. 각각의 이미지 사진을 취득 사진 상자를 클릭합니다. </li><li> 다음, 자동으로 노출 시간을 설정합니다. &quot;픽셀 비닝 또는 CCD 해상도&quot;에서 &quot;보통&quot;을 확인하십시오. 그런 다음 F / 중지하거나 개구에서, 2의 가치를 확인하십시오. 다음, 535 여기 필터와 DsRed 방출 필터를 선택합니다. </li><li> 보기의 분야에서, 이미지 하나의 마우스를보기 B를 클릭하십시오. </li><li> 그런 다음, 이미지 수집을 시작하기 위해 취득을 클릭하십시오. </li><li> 이미지 수집이 완료되면, 관심 지역, 또는 투자 수익 (ROI), 신호의 측정 도구를 사용하십시오. 신호 가치와 영역을 해제하기 위해 측정 아이콘을 클릭합니다. </li><li> 마지막으로, 사용자 폴더에 이미지를 저장하려면 &quot;저장&quot;을 클릭하십시오 </li><li> 이미지가 저장되고 나면, isoflurane의 관리를 중지하고 그 새장에 마우스를 반환합니다. 마우스가 즉시 일어나해야합니다. </li></ol> 5. 대표 결과 - 사용이 프로토콜을, orthotopic 난소암의의 생체내 성장은 비침습 절차를 사용하여 적어도 3 주 동안 모니터링할 수 있습니다. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="그림 1" /> 그림 1. 대조군으로 MOV1 캣 세포, 또는 PBS는 orthotopically. 이외의 종양 발생하기 쉬운 생쥐 (각각 오른쪽과 왼쪽 동물 동물과)의 난소에 주입했다 생체내 이미징에서 2 주 후에 실시되었다. 난소에 engrafted MOV1 캣 세포에 의해 생성된 형광 방출을 측정하고 대조군 마우스에서 해당과 비교했다. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="그림 2" /> 그림 2. MOV1 캣 난소 종양의 냉동 섹션은 mAb가 종양 침투 lymphocytes (검은색 화살표)를 감지하는 DAB 기판 (어두운 갈색) 다음 biotinylated 방지 CD4 물들일되었습니다. 세포는 세포 핵 (파란색)를 시각화하는 메틸 녹색과 counterstained되었습니다. 종양 세포는 (빨간색 화살표) T 세포에서 morphologically 뚜렷한되었습니다. 슬라이드 40x 확대 나타납니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

수술과 주사 Orthotopic 난소 부르사에서 Orthotopic 주입은 교육 및 정밀도를 요구합니다. 그러므로 <ol><li> 첫번째 시신과 함께 가난한 수술 경험, 연습의 경우. </li><li> 그들이 nulliparous 여성과 비교 주입 및 증가 생존을 용이하게 시간이 지남에 따라 더 큰 난소 개발에 우선적으로 multiparous 암컷을 (하나 또는 두 개의 새끼)를 사용합니다. </li><li> 마우스 난소 부르사의 작은 크기?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

배경 : 난소암은 일반적으로 케이스 / 사망자 비율이 높은 고급 단계에서 진단하기 때문에 미국 여성들 사이 1,2,3 모든 gynecologic malignancies의 가장 치명적인 유적이다. 장액의 종양은 난소암의 가장 광범위한 형태이며, 4,5 TG - MISIIR 태그 유전자 변형은 저절로 종양의 유형을 개발하는 유일한 마우스 모델을 나타냅니다. Mullerian 억제 물질 유형 II 수용체 (MISIIR) 유전자 프로 모터 6 제어하에 TG - MISIIR 태그 마우스 표현 SV40 변환 영역. 추가 형질 라인은 그 표현 SV40 태그 transgene을 확인하고 있지만, 난소 종양을 개발하지 않습니다. 비 - 종양 발생하기 쉬운 쥐들이 C57Bl / 6 마​​우스에 대한 일반적인 수명을 전시하고 비옥한 있습니다. 이 생쥐는 TG - MISIIR 태그 - DR26 생쥐에서 분리된 종양 세포에 대한 syngeneic allograft받는 사람으로 사용할 수 있습니다. 목표 : 종양 이미징 7 가능하지만, 깊은 종양의 조기 발견은 작은 생활 동물에 도전합니다. 장액의 난소암에 대한 immunologically 손상 동물 모델의 잠복기 연구를 활성화하기 위해, 우리는 생체내 이미징, 종양 microenvironment 및 종양 면역 반응의 연구에 허가 난소암의 유형에 대한 syngeneic 마우스 모델을 설명합니다. 방법 : 우리는 먼저 26주 된 DR26 TG - MISIIR 태그의 여성에 수확 자연 난소 종양에서 태그 + 마우스 암 세포주 (MOV1)을 파생. 그렇다면, 우리는 안정 Katushka을 인코딩 TurboFP635 Lentivirus 포유 동물 벡터, 여기 / 635분의 588 nm의 8,9,10에서 방출 맥시멈와 말미잘 Entacmaea quadricolor에서 적색 형광 단백질의 멀리 붉은 돌연변와 MOV1 세포 transduced. 우리는 orthotopically 아닌 종양이 발생하기 쉬운 TG - MISIIR 태그 여성 생쥐의 난소 11,12,13,14에서 MOV1 언니를 심었을 거예요. 종양 진행은 생체내 광학 이미징 및 종양의 microenvironment에 immunohistochemistry에 의해 분석되었다 다음되었다. 결과 : Orthotopically MOV1 캣 세포를 이식은 묽은 난소 종양을 개발했습니다. MOV1 캣 종양은 인간의 난소 암 15 (그림 2)에서 관찰, 최대 주입 후 3 주 (그림 1) 생체내 이미징에 의해 시각 될 수 있으며, leukocytes로 가득 차 있었다. 결론 : 우리는 깊은 조직의 산도 안정 높이 Katushka의 photostability에 의한 초기 종양의 생체내 이미징에 적합한 난소암의 orthotopic 모델을 설명합니다. 우리는 종양 면역 반응과 immunotherapies의 생체내 이미징 연구 및 모니터링을위한 장액의 난소암이 소설 syngeneic 모델의 사용을 제안합니다.

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

공부하기<em&gt; 생체내에</em&gt; 종양의 성장과 종양의 microenvironment, 우리는 면역 적격 동물 난소암의 syngeneic 및 orthotopic 마우스 모델을 사용합니다. 우리는 Katushka 형광 단백질 (MOV1와 마우스 종양 세포 라인 (MOV1)를 transduced<sup&gt; KAT</sup&gt;)와 우리가 난소 년에 orthotopic 주입을 표시하고<em&gt; 생체내에</em&gt; 이미지.

에 대한 면역 적격 마우스에 묽은 난소암의 Orthotopic 모델 생체내에</em종양 면역 응답> 종양 이미징 및 모니터링

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K 조회수.  University of Pennsylvania-School of Medicine. 공부하기 생체내에 종양의 성장과 종양의 microenvironment, 우리는 면역 적격 동물 난소암의 syngeneic 및 orthotopic 마우스 모델을 사용합니다. 우리는 Katushka 형광 단백질 (MOV1와 마우스 종양 세포 라인 (MOV1)를 transduced KAT)와 우리가 난소 년에 orthotopic 주입을 표시하고 생체내에 이미지.

비디오: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

연구

면역학 및 감염병학

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8 transiently Transgenized 마우스 모델<em&gt; In Vivo</em&gt; 염증 반응의 장기 모니터링

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Thymus 종속 및 독립 Thymus 면역 글로불린 Isotype 반응 효소 연결 된 Immunosorbent 분석 결과 사용 하 여 마우스의 특성

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

종양 특이 CD8+ T 세포 반응에서 면역 우세를 연구 하기 위한 생체 내 세포 독성 분석 법

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

심질 마우스에 있는 인간 흉막 중피종의 정형외 모형의 PET/CT에 의한 이식 및 모니터링

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

종양 미세 환경에서 면역 세포 집단의 시각화, 정량화 및 매핑

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

마우스에서 신경 면역 역학의 생체 내 이미징에서 반복적인 능력을 발휘하는 정밀한 두뇌 매핑

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

in vitro 종양 미세환경에서 면역 반응을 해부하기 위한 미세유체 공동 배양 모델

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

에 대한 면역 적격 마우스에 묽은 난소암의 Orthotopic 모델 생체내에 종양 이미징 및 모니터링