NKS is supported by NIH award K08 AI108690.

모든 연구는 실험 동물 과학을위한 미국 협회 (AALAS)에 의해 설정된 수의 기준을 충족 하버드 의과 대학에서 기관 동물 관리 및 사용위원회 (IACUC)에 따라 엄격한 검토 및 지침에 따라 실시 하였다. 공동 주택을 통해 박테리아의 1 수평 전송 (옵션) <ol><li> 식품 모두 멸균 된 물을 사용하여 멸균 일회용 케이지를 조립하는 동안 (무균 마우스를 사용하는 경우 특히) 외인성 오염을 최소화하기 위해 무균 기술을 연습. </li><li> 사용 귀 펀치 또는 태그 개별적으로 다른 microbiotas 항구와 같은 케이지에서 그들을 집 6 주령 C57BL / 6 마​​우스를 표시합니다. 마우스 coprophagic하고 케이지 바닥에서 배설물 알약을 먹고 주어진 것으로, 미생물이 자연적으로 마우스 사이에 수평으로 전송됩니다. </li><li> ≥1 주 동안 공동 주택 마우스는 이전의 분석에 미생물 전송 및 면역 학적 변화를위한 시간을 허용합니다. </li></올&gt; 작은 창자 상피내 층과 얇은 판의 propria에서 단일 세포 현탁액 2. 준비 <ol><li> 솔루션의 준비 <ol><li> (소장 당) 추출 미디어 준비 : 30 ml의 RPMI + 93 ㎕의 5 % 디티 오 트레이 톨 (DTT) (/ V w) + 60 ㎕의 0.5 M EDTA + 500 ㎕의 우 태아 혈청 (FBS). 사용하기 전에 즉시 DTT를 추가합니다. </li><li> 25 ml의 RPMI + 12.5 mg을 디스 파제 + 37.5 mg의 콜라게나 II + 300 μL의 FBS (소장 당) 소화 매체를 준비합니다. 사용하기 전에 즉시 디스 파제와 콜라게나 제를 추가합니다. </li><li> 37 ° C에 37 ° C, prewarm 솔루션에서 수행 된 모든 배양하십시오. </li></ol></li><li> 자궁 경부 전위 다음 CO 2 질식하여 마우스를 안락사. </li><li> 아래로 마우스 등의 측면을 놓고 70 % 에탄올로 복부를 스프레이. 순차적으로 이리저리 복부 중간 선을 따라 피부와 후 복막 근막을 절단하여 개복술을 수행하기 위해 가위를 사용하여따라서 복강을 노출, 칼 모양의 과정에 치골을 해요. </li><li> 유문 괄약근에 병행하여 위장에서 소장을 분리하는 가위를 사용합니다. 조심스럽게 장간막 지방을 멀리 괴롭 히고, 복막에서 소장을 제거합니다. <ol><li> 완전히 소장을 제거하려면 ileo-맹장 접합에서 두 번째 컷을 확인합니다. 감기에 고립 소장을 배치 (예., 4 ° C) 10 % FBS를 포함하는 RPMI 세포 생존 능력을 극대화 할 수 있습니다. </li></ol></li><li> 부드럽게 곡선 집게를 사용하여 지방의 큰 조각을 제거합니다. 지방을 제거하는 동안 장 조직 자체를 찢어지지 않도록주의하십시오. </li><li> 주사기에 부착 된 18 G 공급 바늘을 사용하여 차가운 PBS의 20 ㎖ - 15 장 내용, 부드럽게 세척 장을 제거하십시오. </li><li> 보호 프로파일을 절제 가위를 사용하여, 5 % FBS를 포함하는 차가운 RPMI에 배치합니다. 보호 프로파일은 소장의 antimesenteric 측에 위치하며, 멀티 소엽 흰색으로 표시된다질량. 12 프로파일 - 특정 균주에 따라 마우스는 일반적으로 8있다. </li><li> 4 인치 세그먼트 - (3)에 소장을 잘라. </li><li> 멀리 조직에서 지방을 애타게 무딘 메스를 사용하여 RPMI에 적신 종이 타월에 각각의 작은 창자 세그먼트를 압연에 의해 잔여 지방을 제거합니다. </li><li> 곡선 집게로 장 세그먼트를 cannulating 및 조직의 말단부를 파악하여 내부 조직을 켭니다. 부드럽게 조직이 반전되는 결과 (근위 단부에서 시작하는) 곡선으로부터 집게 조직 세그먼트를 제거하기 위해 직선 겸자 쌍을 사용한다. </li><li> 30 추출 미디어 ㎖의 교반 막대가 들어있는 컵에 조직 세그먼트를 배치합니다. 컵에 뚜껑을 고정하고, 37 ℃에서 15 분 동안 500 rpm으로 교반; 교반 활발하지만 난류되지해야합니다. </li><li> 배양 후, 흐린 나타납니다 상피 함유 상층 액에서 조직 조각을 분리 강철 여과기를 사용합니다. 이 뜨는 내가 포함되어 있습니다2.23 - 2.18 단계에서 샘플을 여과로 진행하여, 필요하다면 추가로 분석 될 수 ntraepithelial 림프구. </li><li> 수동 잔류 추출 매체를 씻어 RPMI에서 조직 조각을 선동. </li><li> 건조 종이 타월에 조직을 배치하고이 추출 매질에 의해 해방되지 않은 잔류 점액 제거를 용이하게하기 위해 단부에 걸쳐 여러 번 종료 플립. </li><li> 소화 매체의 600 μL와 1.5 ML 튜브에 조직 조각을 놓습니다. </li><li> 조각은 더 이상 가위에 충실하지 않고 용액이 균질 나타날 때까지 조직을 말하다 가위를 사용합니다. 이 단계에서는 조직 전체 효소 소화를 위해 중요하다. </li><li> 소화 매체의 25 ml에 들어있는 컵에 다진 소장을 추가합니다. 37 ° C에서 30 분 동안 500 rpm에서 교반한다. 반쯤 소화를 통해 (예. 15 분에), 최대 피펫 및 혈청 학적 피펫과 아래 조직의 큰 덩어리를 분쇄하기 위해. </li><li> 필터 소화 조직 (또는 epith단계 2.12에서 elial 층의 경우 50 ML 튜브에 100 μm의 셀 스트레이너를 통해 처리 IELs는). 10 % FBS를 포함하는 RPMI 20 ㎖와 스트레이너를 씻어. </li><li> 4 ℃에서 10 분 동안 500 XG에 필터링 솔루션을 원심 분리기. </li><li> 조심스럽게 10 % FBS를 포함하는 RPMI 1 ml의 상층 액과에 resuspend 펠렛을 가만히 따르다. </li><li> 필터는 50 ㎖ 튜브에 40 μm의 셀 스트레이너를 통해 세포를 재현 탁. 10 % FBS를 포함하는 RPMI 20 ml의 스트레이너를 씻어. </li><li> 원심 분리기는 4 ° C에서 10 분 동안 500 XG에 솔루션을 여과. </li><li> 조심스럽게 2 % FBS를 함유하는 RPMI 1 ml의 상층 액 및 펠렛을 재현 탁 가만히 따르다. </li></ol> 파이어 패치에서 단일 세포 현탁액 3. 준비 <ol><li> 전송 (25) 소화 미디어 ㎖의 교반 막대와 컵에 보호 프로파일을 절제. 보안 뚜껑, 37 ° C에서 10 분 동안 500 rpm에서 스핀. </li><li> 필터는 50 ㎖ 튜브에 40 μm의 셀 스트레이너를 통해 프로파일 소화. 어떤 덩어리의 REMA 경우에, 1 ML의 주사기에서 플런저의 평평한 끝 부분을 사용하여 스트레이너를 통해 키를 누릅니다. </li><li> 10 % FBS를 포함하는 RPMI의 10 ml의 스트레이너를 씻어. </li><li> 원심 분리기는 4 ° C에서 10 분 동안 500 XG에 솔루션을 여과. </li><li> 조심스럽게 2 % FBS를 함유하는 RPMI 1 ml의 상층 액 및 펠렛을 재현 탁 가만히 따르다. </li></ol> 유동 세포 계측법 4. 표면 얼룩 <ol><li> 세포 분취 적절한 부피 (예., LPLS 200 μL)를 96 웰 둥근 바닥 플레이트에. </li><li> 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 접시를 반전시켜 상층 액을 가만히 따르다. </li><li> RPMI이 2 % FBS가 포함 된 90 μL에 재현 탁 세포와 1 : 안티 CD16 / 32 (FC 블록)의 100 희석. 4 ℃에서 10 분 동안 품어. </li><li> 10 μl의 PBS를 추가합니다. 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 접시를 반전시켜 상층 액을 가만히 따르다. </li><li> 250 ㎕의 PBS에 재현 탁 세포. 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 로 가만히 따르다 뜨는반전 판. </li><li> 제조 업체의 프로토콜에 따라, 원하는 경우 생존 염료 (선택 사항) 얼룩 세포. </li><li> 100 ㎕의 1 % 포르말린에 재현 탁 세포는 세포를 해결합니다. 4 ℃에서 어둠 속에서 1 시간 동안 품어. </li><li> 200 μl의 PBS를 추가합니다. 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 접시를 반전시켜 상층 액을 가만히 따르다. </li><li> 250 ㎕의 PBS에 재현 탁 세포. 4 ℃에서 5 분 동안 500 XG에 원심 분리기. 접시를 반전시켜 상층 액을 가만히 따르다. </li><li> 200 ㎕의 PBS에 재현 탁 세포. 유동 세포 계측기를 사용하여 세포를 분석한다. </li></ol>

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No conflicts of interest declared.

우리는 분리를위한 프로토콜을 제시하고 보호 프로파일의 LPLS, IELs 및 림프구를 포함한 작은 장 림프구의 세포 계측 특성 흐름. 미생물의 변화가 작은 창자의 면역 체계에 미치는 영향을 평가에 관심있는 경우, 우리는 세부 사항 다른 microbiotas을 품고 마우스와 생물의 수평 전송에 관련된 간단한 단계. 이 프로토콜은 소장에 초점을 맞추고 있지만, 순서는 오직 차이가없는 프로파일 제거되지한다 (...

소장의 주요 작업은 종종 영양 (1)의 분해 흡수가되는 것으로 간주된다. 대사 기능이 명확 필수적이지만, 소장의 루멘 내에있는 환경이 항원의 연속 사격으로부터 숙주를 보호 동등하게 중요한 역할을한다. 장내 외부 세계를 분리 (예., 내강 항원) 단일 전지 층 두께 상피 층 호스트의 내부 환경에서. 이와 같이, 작은 창자의 면역 시스템은 침입 병원균에 대한 강력한 대응을 설치하는 동안 최소한의와 점막을 입력식이 요법과 공생 미생물에서 외국 항원을 수있는 경우, 면역 반응을 반응성에 대한 임계 값을 균형의 강력한 작업이 다른 &quot;유해한&quot;항원. 이러한 항원에 대한 과도하거나 부적절한 면역 반응은 (병리학 적 질환으로 이어질 수 있습니다 예를 들어,., inflamma토리 장 질환, I 형 당뇨병, 다발성 경화증)과 3-6을 피해야한다. 전반적으로, 위장관 모든 항체 - 분비 세포 (7)의 70 % 이상 함유, 신체에서 가장 큰 면역 기관을 나타낼 것으로 생각된다. 고유 판 (LP)에서, 상피내 층 및 파이어 패치 (PPS) - - 작은 장 면역 체계는 3 개 주요 구획으로 구성되어 각각이 림프구의 독특한 그룹을 포함하는 것이다. LP에서 림프구 (LPLS)은 ~ 20 %의 B 세포 주로 TCRαβ + T 세포이고; 상피내 림프구 (IELs)는 TCRαβ + T 세포보다 TCRγδ + T 세포와 B 극소수의 셀을 포함하고; 그리고 작은 창자 벽에 내장 된 차 림프 기관이다 보호 프로파일은 ~ 8​​0 %의 B 세포가 포함되어 있습니다. 이러한 해부학 적 영역의 각이 약간 별개의 기능과 존재 론적 기반을 가지고 있지만, 그들은에서 작동 아armonized 패션 병원성 모욕에서 호스트를 보호 할 수 있습니다. 또한, 미생물은 특정 미생물, 특히 세포 계통 8,9의 개체 발생의 동족 관계의 인식이 증가함에 따라 장 면역계의 개발을위한 중요한 결정이라고 성장 하락이있다. 또한, 장 면역계의 교육 해부학 먼 부위에 대한 면역 반응에 영향을 미치는 것으로 특정 (예., 관절염, 다발성 경화증, 폐렴), 그 이전에 10 인식보다는 장 면역계의 개발은 더욱 질병 과정에 중요한 것으로 명백 해졌다 -12. 이와 같이 정량적으로 장내 면역 체계를 평가에 대한 관심이 지금 호스트 공생 상호 작용과 많은 전신 질환의 발병도를 포함하는 호스트 병원체 상호 작용 이상으로 확장했다. 현재 방법의 가변성을 감안장내 림프구 분리, 필요한 시간 밸런싱 점점 중요하면서 수율 가능성 및 일관성을 최적화하는 방법. 퍼콜 구배를 포함 프로토콜, 시간과 노동 집약적과 인간의 오류 가능성이 더 많은 경향이 변수 수율로 이어지는 13을 생존. 여기서, 우리는 3 작은 창자의 면역 구획에서 림프구의 분리 및 특성에 최적화 된 프로토콜을 제공합니다. 또한, 점막 면역 체계에서 미생물에 의한 변화에 주어진 관심이 높아지고, 우리는 이러한 변화가 정량적으로 장내 면역 체계에 미치는 영향을 평가하기 위해 마우스와 미생물의 수평 전송을 허용하는 데 사용할 수있는 단계를 포함한다.

<table><tbody><tr><td>Sterile Gloves</td><td>Kimberly-Clark</td><td>555092</td><td></td></tr><tr><td>sterile mouse cage</td><td>Innovive</td><td>MS2-AD</td><td>contains lid, cage bottom, and alpha-dri bedding</td></tr><tr><td>Metal feeder</td><td>Innovive</td><td>M-FEED</td><td></td></tr><tr><td>Water bottle</td><td>Innovive</td><td>M-WB-300</td><td></td></tr><tr><td>Card holder</td><td>Innovive</td><td>CRD-HLD-H</td><td></td></tr><tr><td>Autoclavable rodent chow (NIH-31M)</td><td>Zeigler</td><td>4131207530</td><td></td></tr><tr><td>RPMI medium 1640</td><td>Gibco</td><td>11875-119</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>0.5 M EDTA (pH 8.0)</td><td>Ambion</td><td>AM9262</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GemBio</td><td>100-510</td><td></td></tr><tr><td>Dispase II</td><td>Invitrogen</td><td>17105-041</td><td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td></tr><tr><td>Collagenase, type II&amp;nbsp;</td><td>Invitrogen</td><td>17101-015</td><td>the concentration in the protocol is based on an activity level of 245 U/mg</td></tr><tr><td>Dissecting scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Feeding needle (18 G, 2&quot; length)</td><td>Roboz</td><td>FN-7905</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>305482</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Disposable Scalpel (15 blade)</td><td>Miltex</td><td>4-415</td><td></td></tr><tr><td>Curved forceps</td><td>Roboz</td><td>RS-5211</td><td></td></tr><tr><td>Straight forceps</td><td>Roboz</td><td>RS-5132</td><td></td></tr><tr><td>Multi-purpose cups, 120 ml</td><td>VWR</td><td>89009-662</td><td></td></tr><tr><td>Stir bar</td><td>VWR</td><td>58949-062</td><td></td></tr><tr><td>Multi-position stir plate, 9-position</td><td>VWR</td><td>12621-048</td><td></td></tr><tr><td>Stainless steel conical strainer, 3 inch&amp;nbsp;</td><td>RSVP</td><td></td><td></td></tr><tr><td>1.5 ml Tube</td><td>Eppendorf</td><td>0030 125.150</td><td></td></tr><tr><td>100 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-19</td><td></td></tr><tr><td>40 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-1</td><td></td></tr><tr><td>50 ml Conical tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>96-well Plate, round-bottom</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>Anti-mouse CD16/32 (Fc block)</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>(Optional) Fixable viability dye eFluor 780</td><td>eBiosciences</td><td>65-0865-18</td><td></td></tr><tr><td>10% Formalin, neutral buffered</td><td>Thermo Scientific</td><td>5725</td><td></td></tr></tbody></table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

창자 - 신체의 모든 장기의 면역 세포의 최대 개수를 포함 - 끊임없이 외래 항원, 미생물 및식이 모두 노출된다. 소장은 전신 질환의 숫자에 중요한 내에서 이러한 내강 항원이 면역 반응과 면역 세포의 교육을 형성하는 데 도움 것을 증가 이해 감안할 때, 장내 면역 체계의 특성에 대한 관심이 증가하고있다. 그러나, 많은 문서 프로토콜 힘들고 시간 소모적이다. 우리는 빠른 재현하고 힘든 퍼콜 구배를 필요로하지 않는 소형 장내 점막 고유 층, 상피내 층 및 파이어 패치에서 림프구 분리 여기 간단한 프로토콜을 제시한다. 프로토콜 소장에 초점을 맞추고 있지만, 또한 결장의 분석에 적합하다. 또한, 우리는 특정 과학 QUES에 따라 추가 최적화를해야 할 몇 가지 측면을 강조기. 이 방법은이어서 유세포 분석 또는 특성화의 대체 수단을 사용할 수 림프구 가능한 다수의 분리가 발생.

비장 (도 1a 및도 1b)와 같은 유사한 순방향 및 측면 산란 특성을 갖는 세포 분리 집단을 수득한다 작은 창자 림프구의 단일 세포 현탁액의 세포 계측 분석을 흐른다. 조직은 분리의 초기 단계에서 4 ℃로 유지되지 않은 경우, 림프구는 하부 전방 산란을 갖는 다른 상피 죽은 세포로부터 분리하기 어렵 인 림프구 집단에서 얻어진 다이 시작할 수있다 (도 1C)...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

isolation-flow-cytometric-characterization-murine-small-intestinal

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JoVE Journal

Immunology and Infection

27.3K 조회수.  Boston Children’s Hospital. There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. 

비디오: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

연구

면역학 및 감염병학

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

분리 및 쥐 림프구의 활성화

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

마우스 작은 장의 면역 시스템에서 세포를 분리

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Murine Thymus, 림프절, 비장 Cytometry 사용 하 여 배출 췌 장 하위 집합 규제 T 세포의 결정

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

뮤린 B 세포 발달의 유동 세포 측정 특성화

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

유동 세포측정에 의한 분석을 위한 자궁 내 선천 림프구 세포의 격리

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

뮤린 소장 상피 오가노이드와 선천적 림프성 세포의 공동 배양

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

생물학

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.