이 작품은 신경 질환 및 뇌졸중 (NIH / NINDS) MGC에 R01-NS074387, R01-NS057711 및 R21-NS091555 부여의 / 국립 연구소 건강의 국립 연구소에 의해 지원되었다; NIH / NINDS는 PRL에 R01-NS061107, R01-NS076991, R01-NS082311 및 R21-NS084275을 부여; 레아의 행복한 마음에서 보조금, MGC 및 PRL에게 수여 미시간 종합 암 센터의 대학; 신경 외과, 의학 미시간 대학의 대학의 부; NIH 보조금 2UL1-TR000433 지원하는 임상에 대한 미시간 연구소 및 건강 연구,; NIH / NCI (국립 암 연구소) 부여 T32-CA009676에 의해 지원되는 미시간 대학 암 생물학 교육 보조금; NIH / NINDS 지원하는 임상과 기초 신경 과학 미시간 교육 대학은 T32-NS007222을 부여; 그리고 NIH / NIGMS (일반 의학 과학의 국립 연구소)에 의해 지원 미시간 의료 과학자 교육 프로그램의 대학은 T32-GM007863을 부여합니다. 저자박사 카린 Muraszko 및 신경 외과의 부서에서받은 교육 리더십과 지원에 감사 재; M. 달 그렌, D. Tomford,과 최상의 행정 지원에 대한 S. 나폴리탄에; 뛰어난 기술 지원 M. Dzaman에; 그리고 자이스 3D 스캐닝 전자 현미경의 구입을 향해 관대 한 지원을위한 필 F. 젠킨스에. 우리는 또한 뇌 단핵 세포의 분리를위한 밀도 원심 분리 미디어 매개 전략의 수정 된 버전이 그려진하는 하버드 의과 대학에서 Kuchroo 실험실을 인정합니다.

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저자가 공개하는 게 없다.

이 프로토콜은 초기 마우스 뇌 종양 미세 환경에 침투 한 한 PBMC의 분리 및 유세포 분석을위한 견고하고 재생 가능한 방법을 설명한다. 신경 교종 세포 현탁액 정위 마우스 뇌의 선조체에 접목 된 실험자에 의해 지정된 농도로 생성된다. 마우스는 다음 실험 디자인에 의해 지정된 소정의 시점에서 안락사 그들의 뇌를 수거하고, 형광 접합 된 일차 항체의 조합으로되어 immunolabeled 교종 함침 한 PBMC?...

신경 교종은 중추 신경계 (CNS)에서 발생하는 변형 신경교 neuroepithelial 뇌암의 클래스이다. 모든 신경 교종의, 세계 보건기구 (WHO) 등급 IV 신경 교종, 또는 아교 모세포종 (GBM), 가장 일반적인 1 치명적이다. GBM은 테모 졸로 마이드 2 방사선 플러스 수반과 보조 화학 요법 다음에 가능한 범위에 종양 절제술로 구성되어 현재의 표준 진료에 매우 내화입니다. 이 치명적인 암 5 년 3 후에 질병을 생존 환자의 5 %와 초기 진단의 시간에서 생존의 15-18개월의 실력 예후를 수행한다. 혈액 뇌 장벽 (BBB)의 존재는, 전문적인 항원 제시 세포의 부족 (장갑차) 및 뇌 내에서 4 선의 림프 구조의 존재는 이전 미확인 특권 면역 GBM의 개념을 이끌어왔다. 그러나, 많은 연구가 지금 SHO이러한 뇌 암은 참으로 단핵구, 대 식세포 및 골수 유래 억제 세포 (MDSCs) (5)를 포함 기원에 주로 골수 있습니다 주변 면역 세포의 채용을 발생시킬 것을 승. GBM은 6,7 프로 종양 될 뇌 상주 미세 아교 세포의 활동에 영향을 미친다. 이러한 CD8 + T 세포 (8)과 CD56 + 자연 살해 세포 (9) 등의 림프 세포는 종양 미세 환경 내 존재하지만 훨씬 적은 숫자 생각 사실 (인해 종양 연관된 대 식세포에 신경 교종 - 유래 요인에 의해 유발 면역 기능 할 TAMS) 10. CD4 + T 세포는 GBM도 존재하지만이 인구의 대부분은 CD25 및 Foxp3를, 면역 T 규제 (T의 REG) 셀 (11)의 업체를 표현한다. GBM의 전반적인 면역 억제 상태는 면역 탈출 및 종양 진행 (12)의 증진에 절정. 13-19을 개발하기 위해 뇌 종양 immunosuppresson의 메커니즘을 극복하기 위해 노력하고있다. 이 작품의 절정은 이제 새로 진단 GBM (: NCT01811992 ClinicalTrials.gov 식별자) 환자 결합 된 세포 독성 및 면역 자극 치료를 평가하기위한 임상 시험을 주도하고있다. 가장 최근의 연구는 마우스 GL26 쥐 CNS -1- GBM 셀 β - 갈 락토 시드 - 결합 렉틴 ​​galectin-1 (GAL-1) ~ (20)의 대량 생산함으로써 항암 NK 세포의 면역 감시를 차단하는 것으로 나타났다. 이것은 shRNA를 매개 유전자 녹다운을 사용하여 신경 교종 세포의 GAL-1의 발현을 억제함으로써 입증되었다. 시험 관내 experimenTS는 GAL-1 결핍 신경 교종 세포 배양에 정상적으로 증식 것으로 나타났다, 아직 동계 C57BL / 6J 또는 RAG1에 두개 내 생착 후 곧 빠른 거절을 시행 - / - 마우스, 따라서 이러한 형태에 T- 또는 B- 세포의 독립성을 확립 종양 제거. 안티 아시 알로 GM 1 항혈청 또는 GAL-1 결핍 신경 교종 제거에서 NK 세포의 역할을 설정하는 두개 GAL-1 결핍 교종 성장의 완전한 회복되었다 모노클 NK1.1 항체와 NK 세포 immunodepletion. 우리는 지금 GR-1 + /는 CD11b + 골수 세포의 immunodepletion 표시하는 따라서 NK 매개 GAL의 돕는데 골수 세포를위한 필수 불가결 한 보조 역할을 드러내는, NK 세포의 존재에도 불구하고 GAL-1 결핍 신경 교종 제거를 방지하기에 충분한 -1 결핍 종양 용해 (게시되지 않은 데이터). 이는 예기치 않은 결과는 말초 혈 단핵 세포의 분리 및 분석 (한 PBMC)를위한 광범위한 프로토콜을 개발할 주도했음을우리는 더 나은 GAL-1 결핍 신경 교종 거부 조건부 면역 침투 이벤트를 특성화 할 수 있도록 즉시 두개 내 생착 후 뇌 종양 미세 환경에 침투. 방법은 GL26-CIT를 불리는 구조적 익스프레스 mCitrine 형광 단백질은, 형광 현미경 (21)에 의해 직접 종양 세포의 가시화를 허용 GL26 마우스의 신경 교종 세포를 사용하여 여기에 설명된다. 이 세포들은 정위 동계 C57BL / 6J 마우스의 뇌에 접목하고, 이전에 마우스 안락사에 24, 48, 또는 72 시간 동안 성장을 사용할 수 있습니다. 신경 교종-침투 PBMC를 다음 항 -CD45를 사용하여 격리 및 immunolabeled되어, -GR-1, -CD11b과 그랜 자임 B의 세포 내 immunolabeling와 함께 -NK1.1 세포 표면 항체 (GzmB). 항체의 이러한 특정 조합이 우리가 B가 종양 침윤 GR-1 + / + CD11b를 골수 세포의 식별 및 NK1.1 +, NK 세포, 세포 유형을 허용EEN는 GAL-1 결핍 종양 제거에 관여. GAL-1 결핍 GL26-CIT의 신경 교종의 면역 침투 프로파일, GL26-CIT의-gal1i 여기에 언급하고 비를 포함 GAL-1 GL26-CIT의-NT 호출의 정상 수준을 발현하는 신경 교종의 것과 비교된다 제어 shRNA를 머리핀을 대상으로. 프로토콜 동소 동계 C57BL / 6J 마우스의 선조체로 이들 세포를 접목하는 방법에 대한 설명이 뒤 따른다 시험 관내 배양 방법 GL26-CIT의 신경 교종 세포에 대한 설명으로 시작한다. 그러므로 분리 및 유세포 분석 교종 함침 된 PBMC의 immunolabeling 관련된 단계를 열거 진행한다. 표준 프로토콜은 데이터 분석 및 그래픽 표현의 설명으로 종결. 데모는 밝혀 그 모두 GR-1 + /는 CD11b + 골수 세포와 NK1.1 + NK 세포 우선적으로 48 내 여자-1 결핍 뇌 종양 미세 환경 내에서 축적종양 주입,이 종양이 빠른 속도로 완전한 종양 용해 약 일주 후 종양 생착 (20)를 받아야하는 이유를 설명하는 데 도움이되는 결과의 시간. 상기 방법은 뇌에 면역 침윤 시간에 데이터를 필요로하는 생체 내 다양한 실험 디자인에 용이하게 적응할 수있다. 실험자들은 단일 분석 될 샘플의 수에 따라 약 4-6 시간에 신경 교종 함침 된 PBMC의 세포 계측 분석을 흐르게 뇌 적출로부터 프로토콜을 수행 할 수있다. 방법은 또한 특히 종양 미세 환경에 의해 유도 된 면역 표현형을 식별하도록 뇌 침투 것들과의 비교를 위해 종양 함유 마우스에서 PBMC를 순환 프로파일을 특성화하기 위해 실험 목적과 결합 될 수있다. 이것과 유사한 방법을 적용 뇌 종양 미세 환경 내로 말초 면역 세포의 거래에 관련된 요인의 더 나은 이해를 용이하게한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td>without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm deep</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% NaCl injection, USP</td>
			<td>Hospira</td>
			<td>NDC: 0409-4888</td>
			<td>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride injection</td>
			<td>Orion Pharma</td>
			<td>NDC: 52483-6298</td>
			<td>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clippers</td>
			<td>3M</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone-iodine solution</td>
			<td>Aplicare</td>
			<td>82-217</td>
			<td>NDC: 52380-1905</td>
		</tr>
		<tr>
			<td>Sterile petrolatum ophthalmic ointment&#160;</td>
			<td>Dechra</td>
			<td>NDC: 17033-211</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol prep pads</td>
			<td>Kendall</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td>non-woven, 4&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml conical polypropylene microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse stereotactic frame</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Philips Burton</td>
			<td>CS316W</td>
			<td>Coolspot II Variable Spotlight</td>
		</tr>
		<tr>
			<td>Curved dissecting forceps</td>
			<td>Ted Pella Inc.</td>
			<td>5431</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri retractors&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</td>
			<td>1100-01</td>
			<td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td>
		</tr>
		<tr>
			<td>Engraving Cutter</td>
			<td>Dremel</td>
			<td>105</td>
			<td>1/32&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L glass screw-cap storage bottle</td>
			<td>Corning</td>
			<td>1395</td>
			<td>Type 1, Class A borosilicate glass</td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/ml solution</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole Parmer Instruments Group</td>
			<td>77200-60</td>
			<td>Master Flex Easy Load II console drive</td>
		</tr>
		<tr>
			<td>compressed carbogen (95% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>from Clostridium histolyticum&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Antistatic polystyrene hexagonal weighing dishes</td>
			<td>Ted Pella Inc.</td>
			<td>20157-3</td>
			<td>top I.D. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</td>
			<td>Garvey</td>
			<td>40475</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone rongeurs</td>
			<td>FST</td>
			<td>16001-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13014-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers&#160;</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td>
			<td>Biolegend</td>
			<td>103128</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td>
			<td>eBiosciences&#160;</td>
			<td>17-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td>
			<td>BD Pharmigen</td>
			<td>553128</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control</td>
			<td>Biolegend</td>
			<td>400632</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Special Order Research Product flow cytometer/cell sorter</td>
			<td>BD&#160;</td>
			<td>650033</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG603</td>
			<td>model: SterilGARD III Advance</td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

우리 연구소는 최근 자연 살해 (NK) 세포가 암 세포가 β - 갈 락토 - 결합 렉틴 galectin의 발현에 결함이 렌더링되는 경우 두개 내 생착 후 CNS-1 악성 신경 교종 곧 동소 이식 마우스 GL26과 쥐를 박멸 할 수 있음을 보여 주었다 -1 (GAL-1). 최근의 연구는 지금 GR-1 + /는 CD11b + 골수 세포의 인구는이 효과에 중요한 것을 알 수있다. 더 NK와 골수 세포가 우리가 단리 교종 함침 말초 혈 단핵 세포 (PBMC)의 분석을위한 광범위한 프로토콜을 개발했다 GAL-1 결핍 종양 거부를 부여하기 위해 협력하는 메커니즘을 이해하기. 이 방법의 종양 미세 환경 내로 침투 PBMC를 비교하여 여기에서 설명된다 shRNA를 녹다운 통해 GAL-1 결핍 렌더링하는 것과 GL26 교종을 GAL-1 발현. 프로토콜에 대한 설명으로 시작하는 방법 문화와 GL26 가전을 준비동계 C57BL / 6J 마우스의 뇌에 접종에 대한 재편. 그러므로 초기 뇌 종양 미세 환경에서 신경 교종 함침 된 PBMC의 분리 및 유세포 분석에 관련된 단계를 설명한다. 상기 방법은 뇌에 면역 침윤 시간에 데이터를 필요로하는 생체 내 실험 디자인에 적용 할 수있다. 교종 함침 된 PBMC는 독립적 인 실험 내내 시점 유사한 종양에서 관찰 유사한 세포 수와 24 시간 후 종양 생착 자마자 두개 내 종양으로부터 분리 될 수있어서, 민감하고 재현성이다. 실험자들은 단일 분석 될 샘플의 수에 따라 약 4-6 시간에 신경 교종 함침 된 PBMC의 세포 계측 분석을 흐르게 뇌 적출에서 방법을 수행 할 수있다. 대안 교종 모델 및 / 또는 셀 특정 검출 항체는 또한 몇몇 다른 immun의 침윤을 평가 experimentalists &#39;판단에 사용될 수있다전체적인 절차 변경 없이도 관심 E 세포 유형.

다음 게이팅 전략은 일반적인 실험에 사용되는 : FSC-SSC-대 → SSC-H 대 SSC-W → FSC-H FSC-W → CD45 대 대 카운트 → GR-1 대 CD11b를 → 카운트 대 NK1.1. 게이트는 GzmB 표현 (도 5a)에 기초하여 층화 GR-1 + / + CD11b를 골수 NK1.1 + 세포 및 NK 세포에 배치. NK1.1 + NK 세포 및 GR-1 + / CD11b를 SSC-A는 NK 세포의 작은 림프 크기 및 골수 세포의 비교적 큰 크기를 확인 대 FSC...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

초기 뇌 종양 미세 환경에 진입 면역 세포의 수와 활성 상태에서 시간에 따른 양적 데이터를 산출 교종 함침 말초 혈액 단핵 세포의 분리 및 유세포 분석을위한 간단한 방법이 여기에서 제시 하였다.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

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JoVE Journal

Immunology and Infection

15.7K 조회수.  University of Michigan.  초기 뇌 종양 미세 환경에 진입 면역 세포의 수와 활성 상태에서 시간에 따른 양적 데이터를 산출 교종 함침 말초 혈액 단핵 세포의 분리 및 유세포 분석을위한 간단한 방법이 여기에서 제시 하였다. 

비디오: Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

마우스 복막 구멍 세포의 분리

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

연구

면역학 및 감염병학

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의​​해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

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 ...

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

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

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

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

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

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스 복막 구멍 세포의 분리

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도

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

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

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

쥐 소장 림프구의 분리 및 유세포 특성

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스 복막 구멍 세포의 분리

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의​​해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도

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

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

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

쥐 소장 림프구의 분리 및 유세포 특성

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

공동 stimulatory 신호 봉쇄와 Alloantigen 자극에 의해 사람 말초혈 Mononuclear 세포 Alloantigen 특정 Anergy의 유도