우리는 트리파노소마 생물학에 대한 일반적인 조언 조지 크로스를 인정하고 싶습니다. 이 작품은 또한 DS, MRM에 NSF 대학원 연구 활동 (DGE-1325261)와 FNP에 대한 NIH / NIAID (허가 번호의 AI085973)에 빌과 멜린다 게이츠 재단 GCE 보조금에 의해 지원되었다. 우리는 I-SCEI 유전자 및 인식 사이트를 포함하는 균주의 사용 갈라드리엘 오두막집 - 광부 감사합니다.

주 : 절차를 통해,이 얼음에 세포를 유지하는 것이 필요하다. 콜드 매체는 또한 전체에 사용되어야한다. 1. 샘플 수확 <ol><li> 100 만 / ㎖ - 0.5의 밀도에 리스터 427 변형 혈류의 trypanosomes에 성장. 이 기생충의 소수 문화를 시작하는 것이 가장 좋습니다. 1500 x g에서 10 분 동안 50 × 106 세포 / 샘플을 스핀. 이상과 같은 긍정적이고 부정적인 항체 컨트롤을 사용하기위한 문화 1 × 10 6 세포를 남겨해야합니다. 참고 :이 프로토콜은 동물의 피 (자세한 내용은 설명을 참조)에서 분리 trypanosomes에 사용할 수 있습니다. </li><li> 피펫 또는 상층 액의 대부분을 부어 (약 750 μl를 떠나). 1.5 ml의 마이크로 원심 튜브에 세포를 전송합니다. </li><li> 미세 원심에서 5,200 XG에서 4 분 동안 4 ° C에서 세포를 스핀과 상층 액을 제거합니다. </li></ol> 2. 자기 라벨링 <ol><li> 를 Resuspend 세포 150 ㎕를 배지에서 (HMI-9 위스콘신적절한 희석 예를 들어 제 혈청) + 기본 안티 VSG 항체. 참고 : 안티 - VSG 항체 집에서 만든 1시 50분의 희석에 사용된다. 이는 형광으로 표지되지 않은 기본 항 VSG 항체를 사용하는 것이 중요하다. </li><li> 10 분 동안 4 ° C에서 추운 방에서 8 - 속도 6에서 소용돌이 어댑터-60을 사용하여 소용돌이 세포. 1000 μl의 차가운 HMI-9 혈청 - 800 씻으십시오. </li><li> 5,200 XG에 4 분 4 ° C에서 원심과 뜨는을 대기음. 혈청 1ml를 HMI-9로 세척하고, 상기 회전. 뜨는을 대기음. 혈청와 HMI-9 100 ㎕의를 Resuspend 펠릿. </li><li> (MSC) 마이크로 비드를 정렬 자기 활성화 셀의 110 μl를 추가합니다. 기본 안티 VSG 항체에 적절한 항 - 마우스, 항 - 토끼, 또는 안티 - 비오틴 구슬을 사용합니다. 잘 섞다. 10 분 동안 4 ° C에서 추운 방에 소용돌이 세포. </li><li> 세포가 텍싱하는 동안, 분리 자석에 하나의 자기 활성화 분리 컬럼 / 샘플을 설정합니다. 재를해야합니다컬럼 아래 ceptacle (우리는 15 mL의 원심 분리 튜브를 사용하여)를 통해 유동을 수집한다. 추운 방에서 장치를 설정합니다. </li><li> 주요 항목에 대한 혈청 HMI-9의 2 ML을 추가합니다. 흐름을 통해 각각의 샘플에서 수집 열에서 15 ML의 원심 분리기 튜브 장소를 프라이밍 후. </li><li> 단계 2.4에서 10 분 부화 후, 800-1,000 μl의 차가운 HMI-9 혈청을 추가합니다. 5,200 XG에 4 분 4 ° C에서 원심과 뜨는을 제거 언 바운드 항체를 제거합니다. 혈청 및 반복 상층 액 100 μl를 떠나와 HMI-9의 1 ㎖로 세척 할 것. </li><li> 적극적으로 시각적으로 원심 분리기 튜브를 가볍게 펠렛 완전히 재현 탁되어 있는지 확인하기 위해 튜브를 검사합니다. 세포가 잘 재현 탁되어 있는지 확인 위아래로 혈청 피펫 HMI-9의 1 ML을 추가합니다. </li></ol> 3. 자기 분리 <ol><li> 상술 한 바와 같이 열 준비가 끝났다되어 있는지 확인합니다. 열에 셀을 적용합니다. 세포를 비 - 지배적 인 VS를 표현하는 관류 모아서컬럼을 통과하기 위해 7 분 - G. 1 매체 ml의 + 트리 파노 솜은 일반적으로 6 걸립니다. </li><li> 워시 1 ml의 HMI-9 혈청과 함께 배와 단계 3.1에서와 같이 흐름을 통해 동일한 튜브를 수집합니다. </li><li> 나누기 관류 ​​(3 mL) 중 두 가지를 1.5 ml의 마이크로 원심 튜브에. 주의 : 이러한 나중에 결합 될 수 있거나 절반 등의 RNA를 분리하는데 사용될 수있다 </li><li> 자석에서 열을 제거합니다. 열에 혈청 HMI-9 3 ㎖를 추가하고 시작, 지배 VSG를 발현하는 세포를 얻기 위해 15 ㎖의 원심 분리기 튜브에 열을 뛰어. 나중에 분석을 위해 용출 물질의 300 μl를 제거합니다. 얼음이 세포 보관하십시오. </li><li> 양성 및 음성 대조군으로 사용하기 위해, 37 ℃에서 성장 대조군 세포의 시작 배양에서 약 1 백만 셀을 1.5 ml의 마이크로 원심 튜브로하고 피펫 (그 자주 전환 할 것으로 예상되지 않음). 참고 : 양성 대조군 세포 단계 4.1에서 형광 표지 항체로 염색됩니다. 대조군 세포 REMA프로토콜의 나머지 부분에 걸쳐 흠이다. </li><li> 스핀 흐름을 통해 샘플 및 5,200 XG에 4 분 4 ° C에서 긍정적 인 항체 제어 샘플 및 상층 액을 제거합니다. 주 : 용출 다른 셀 300 μL 분취 안티 VSG 염색에 대한 양성 대조군으로 사용할 수있다. </li></ol> 4. 염색은 오염 물질 및 스위처를 확인하는 <ol><li> 재현 탁 플로우 스루 샘플 100 ㎕ HMI -9- 적절한 희석 혈청 + 일차, 형광 표지 된, 항 VSG 항체 양성 대조군 세포. 참고 : 표지 항 VSG 항체 집에서 만든 1의 희석에 사용됩니다 : 1000. 형광 표지는, 안티 VSG는 step.There 단계 3.5 다음과 같은 두 가지의 microcentrifuge 튜브의 흐름을 통해 샘플의 3 ㎖ 있어야 MACS 분리에 레이블이없는 항체와 같은 소스에서 할 수있다. </li><li> 다시 SUS 동안 두 마이크로 원심 튜브의 펠렛을 결합, 유동 세포 계측법에 의해 관통 흐름 모두를 분석100 ㎕ HMI-9 세럼 + 안티 VSG 항체의 샘플을 보류. 잘 섞다. </li><li> 10 분 동안 4 ° C에서 추운 방에 소용돌이 세포. 1000 μl의 차가운 HMI-9 혈청 - 800을 추가합니다. 4 분 4 ° C에서 회전 샘플 상층 액 5,200 XG에 제거는 언 바운드 항체를 제거합니다. </li><li> 혈청와 HMI-9의 1 ml의 세포를 씻으십시오. 4 분 4 ° C에서 회전 샘플 상층 액 5,200 XG에 제거합니다. 이 마지막 스핀 동안 단계 3.4과 음성 대조군 시료에서 용출-샘플을 스핀 다운. </li><li> 148.5 μL HMI-9 혈청 25 ㎕를 절대 계산 구슬과 1.5 μL 프로피 디움 요오드 용액 (175 μl의 총을) 염색 : 모든 흐름 통해 용출 샘플을 재현 탁. 175 μL에 긍정적이고 부정적인 항체 제어 샘플을 재현 탁 HMI-9 혈청. 절대 계산 비즈 / 사용되는 특정 많은 25 μL의 수를 기록해야합니다. </li><li> 사이토의 흐름을 통해 모든 샘플을 실행하고 데이터를 수집합니다. PI로 얼룩 죽은 세포 <sup&gt; +. 오염 물질은 PI로 얼룩 - VSG +를. VSG - - 스위처는 PI로 얼룩 8. </li><li> 데이터 유동 세포 계측법을 사용하여 스위칭 주파수를 계산합니다. 주 : 스위칭 주파수를 계산하는 방법에 대한 자세한 내용은 보충 파일을 참조하십시오 <a href="https://www.jove.com/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">유동 세포 계측법 Calculations.docx</a> 표 1. </li></ol>

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The authors have nothing to disclose.

실험 방법에 대하여, 프로토콜의 가장 중요한 요소는 차가운 모든 샘플을 유지하고있다. 트리 파노 솜은 매우 빠르게 그 표면 (9)에 결합 된 항체를 내재화하지만,이 프로세스는 운동성 의존하고, 셀 4 ° C에서 유지되는 한 상기 분석에 영향을 미치지 않는다. 모든 샘플은 항상 얼음에 보관해야하고, 피펫은 25 ° C 랩 환경에의 노출을 최소화하기 위해 신속하게 수행해야합니다. 콜드 HMI -9- ?...

원생 동물 기생충, 트리파노소마 (Trypanosoma) brucei는 사하라 사막 이남의 아프리카 전역 (트리파노소마 (Trypanosoma) brucei brucei를 통해)과 동물 (트리파노소마 (Trypanosoma) brucei의 gambiense 및 트리파노소마 (Trypanosoma) brucei의 rhodesiense를 통해) 인간에 영향을 미치는 질병의 원인이되는 에이전트입니다. 이것은 체체 파리 벡터의 타액을 통해 포유 동물 숙주에 전달된다. 인간과 동물 아프리카 트리파노소마 증은 모두 발병 지역에서 심각한 경제적 부담을 야기하고, 몇 가지 약물을 사용할 수 또는 두 질병을 치료하기 위해 개발에 있습니다. 면역 회피의 메커니즘을 이해하는 것은 트리파노소마 증에 대한 약물의 개발을위한 중요합니다. 기생충을 포함 밀도, 변형 표면 당 단백질 (VSG) 코트의 항원 변화는 주요 수단 중 하나입니다에 의해 T. brucei는 포유류 항체 반응을 기피한다. ~ 15 EXPRES을 VSG 유전자 약 2,000 변형은 트리파노소마 게놈에 존재하지만, 단지 하나의 하나에서 임의의 주어진 시간에 전사되는시온 사이트. 발현 VSG의 &#39;전환&#39;는 활성 발현 사이트에 VSG 유전자를 복사하여, 또는 (1 검토) 이전에 침묵 식 사이트의 전사 활성화에 의해 하나 발생할 수 있습니다. 많은 작업은 T.에 대한 이해 항원 변화에 헌신하고있다지만 brucei, 스위칭 주파수에 영향을 미치는 요인은 여전히 저조한 이해된다. 현재까지 연구 전환 체외에서 확률 적으로 발생하지만, 스위칭 주파수는 약 106 개 세포를 2 일 정도의 매우 낮다는 사실에 의해 저해되었다. 이 전환은 처음에 검출하기 어렵다 이후 소정 계수가 증가하거나, 스위칭 주파수를 감소 여부를 측정하는 것이 곤란한다. 2009 년 이전에, 주어진 인구 스위처를 분리하는 방법은 긴했고, 노동 집약적. 이 포함 된 지배적 인 VSG 예방 접종을 마우스를 통해 트리 파노 솜을 계대 후 수확 세포하루 이상 3, 면역 4,5에 의해 세포의 수백 계산. 약물 내성이 스위처 (6)를 분리하는 또 다른 전략은 선택에 의존한다. 시험 관내에서 재배 아프리카 트리 파노 솜은 일반적으로 하나의 주요 변형 및 대체 변형을 표현 스위처 훨씬 작은 인구를 표현하는 많은 인구로 구성되어 있기 때문에, 우리는 지배, 시작 VSG 본 논문을 통해 주요 변형을 참조하십시오. 이 과정에서 우리는 결코이 주요 변형 인구의 다른 변종보다 큰 체력을 가지고 있다는 것을 의미하고 싶습니다. 4 시간 - 여기를 확실하게 3에 주어진 집단에서 비 우세 VSG를 나타내는 트리 파노 솜의 개수를 측정 할 수있는 방법을 설명한다. 이 방법은 하나의 주어진 유전자 조작 또는 약물 치료 집단을 전환 세포의 수를 증가 여부를 확인하고자 할 때 특히 유용하다. 대신에 할 일을 발현하는 세포의 인구를 면하게의minant는 약물을 통해 VSG를 시작하거나 면역에 의해,이 세포는 먼저 자기 열을 분리 한 다음 지배적 VSG에 대해 항체에 결합 자석 구슬로 코팅에 의해 제거된다. 스위치드 인구는 플로우를 통해 수집하고, 오염 물질을 식별하기위한 형광 표지 항 VSG 항체 다시 염색된다. 정량 세포 구슬의 비를 구한 인구 7 스위처 수를 정량화하는데 사용될 수 있도록 각각의 샘플 절대 계수 비드 정의 번호를 추가함으로써 달성된다.

<table border="0" cellpadding="0" cellspacing="0" width="644">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">unlabeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fluorophore labeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HMI-9 media</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">HMI-9 is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propidium Iodide</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CountBright absolute counting beads</td>
			<td style="width:143px;">Thermofisher Scientific</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LD columns</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnetic separator</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">vortex adapter-60</td>
			<td style="width:143px;">ThermoFisher scientific</td>
			<td>AM10014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flow cytometer</td>
			<td style="width:143px;">Coulter</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">flow cytometer analysis software</td>
			<td style="width:143px;">FloJo</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

여기에 기재된 방법은 인구 8 스위처의 수를 증가 VSG 발현 사이트 내에서 해당 이중 가닥 나누기를 입증 하였다. 여기서는 마찬가지로 발현 부위에 이중 가닥 브레이크를 생성하도록 유도 된 트리 파노 솜의 인구에서 대표적인 결과를 나타낸다. 우리는 이중 가닥 휴식을 생성하는 유도되지 않은 것과 이러한 trypanosomes에 비교. 1은 흐름을 통해 자기 활?...

Watch this Scientific Journal Video about Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry at JoVE.com

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

검출<em&gt; 트리파노소마 (Trypanosoma) brucei</em자기 활성 셀 정렬 및 유동 세포 계측법에 의해&gt; 변형 표면 당 단백질 전환

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

detection-trypanosoma-brucei-variant-surface-glycoprotein-switching

Research

JoVE Journal

Immunology and Infection

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

8.7K 조회수.  Rockefeller University. African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

비디오: Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

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

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

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

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

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

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Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

Telomere G - 오버행 구조의 시험 Trypanosoma brucei</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

높은 처리량 유전자 태그에<em&gt; 트리파노소마 (Trypanosoma) brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

유동 세포 계측법에 의해 쥐의 신장에서 대 식세포의 표현형 특성

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

이미징 유동 세포 계측법을 사용하여 T 세포 Alloreactivity의 측정

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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