我们承认，从国家健康与医学研究委员会的资金支持（授予APP605524，490037和1047082），澳大利亚研究理事会（批DP12010061），澳大利亚从创新系国家合作研究基础设施战略和教育投资基金，产业，科学和研究。 PML是澳大利亚研究生奖的获得者。

所有的程序均按照麦考瑞大学的政策进行，符合国家卫生和医学研究委员会（NHMRC）实践澳洲代码。根据协议道德没有ARA 2012/017批准，并从动物伦理委员会在麦考瑞大学获得进行这项工作。所有实验均在SJL / J小鼠进行，除非另有说明。 1.鼠标和实验疟疾感染<ol><li>家鼠控制温度（21°C）具有12:12小时明暗周期下。 </li><li>解冻冷冻保存P的等分试样chabaudi阿达米 DS用5％寄生红细胞（pRBCs）注入200微升进入腹腔的C57BL / 6小鼠供体腔。 </li><li>使用流式细胞仪在第3节中描述2天 - - 本协议的4监察捐助鼠标原虫每1。一旦捐助者达到5 - 15％原虫，通过心脏穿刺采血如下： <ol><li>吸100微升肝素溶液（300 U / ml肝素在小鼠林格液（MTR）（154毫米氯化钠，5.6毫米氯化钾，1毫米氯化镁2，2.2毫米氯化钙2，20毫米的HEPES，10毫米的葡萄糖，30 U / ml的肝素，pH值7.4，0.22微米过滤灭菌））到一个1ml注射器用26ģ针。 </li><li>麻醉小鼠通过吸入，用5％异氟烷，由缺乏踏板戒断反应及角膜反射确认麻醉的所需深度。 </li><li>放置鼠标在其一侧并垂直插入针的正下方的弯头，通过肋，并进入心脏。慢慢拉出注射器活塞和0.5，直到旋转针 - 1毫升血液中获得。 </li><li>通过颈脱位麻醉下执行人道的安乐死。 </li></ol></li><li>计算每个供体血容量pRBCs假设的9×10 6 RBC /μL（血液计数，即，如果捐助者是在10％原虫牺牲时，血液将包含9×10 5猪红细胞/微升）。稀释血液寄生于Krebs缓冲盐水（126 mM氯化钠，2.5mM的氯化钾，25mM的碳酸氢钠 ，1.2毫的NaH 2 PO 4，1.2毫的MgCl 2，2.5mM的氯化钙 ，0.2％葡萄糖，pH为7.2，灭菌0.22μm的过滤器）使浓度为每200微升1×10 4个pRBCs并注入200μl的进腹腔所需红细胞标签和输血实验小鼠的空腔。 </li><li>使用流式细胞仪在第3描述2天 - - 4本协议的监控原虫每1。 </li></ol> 2.标签红细胞和输血<ol><li>如在步骤1.3中描述收集来自小鼠的肝素化血液通过心脏穿刺，并放置在冰上。保持血液在4℃下在任何时候。收集每只小鼠约200微升的血液被注入。如果需要的话，分离血成两个样品，并根据需要处理一个样本。 注：以这种方式，侵入经处理的SAMP的乐可以比作对照，未处理的样品（ 见图1的原理图）。 </li><li>制备各荧光红细胞标签的2倍的溶液。 <ol><li>稀阿托633-N-羟基（阿托633-NHS）在地铁的2毫克/毫升的储备溶液，使其浓度为20微克/毫升。 </li><li>稀释生物素-N-羟基琥珀酰亚胺（生物素NHS）在地铁的25毫克/毫升的储备溶液，使其浓度为250微克/毫升。 </li></ol></li><li>拆分血样成两个管和2倍阿托633-NHS溶液等体积添加到一个管中，与2倍生物素 - NHS等体积的其他管。立即混合。孵育血液在4℃下1小时以恒定缓慢混合，或可替代地，混合样品每10 - 15分钟。 </li><li>与地铁（MTR + 0.5％牛血清白蛋白（BSA），灭菌0.22μm滤器）洗涤标记的血液的3倍，如下所示： <ol><li>至少添加2体积地铁，离心机在750×g离心5分钟，去除上清，并resuspend。在至少2卷地铁沉淀。重复2次以上。 </li><li>在最后的洗涤，离心，在750×g离心5分钟，并结合后以不同的组合的沉淀血液（ 即，未处理的阿托633 /治疗生物素，未处理的生物素/处理阿托633）。加入地铁弥补对每只小鼠200微升体积被注入。 </li></ol></li><li>注入标记的血液进入血管内灭鼠疟疾感染的小鼠或未受感染的控制如下： <ol><li>在寄生虫裂殖生殖高峰15％原虫 - 执行输血2。 注：本约一半是通过暗周期（ 即在12 -上午02时在一个正常的光照周期，或12 -下午2点在反向光周期）。 </li><li>用热灯5-10分钟暖小鼠增加血流量，但须小心不要过热的小鼠。小鼠放置在约束装置和血管内注入200微升标记的血液（约1 - 2×10 9 RBCS）通过尾静脉。 </li></ol></li><li>如在第3节中所述采集血样，在注射后不同的时间点。 注：侵入率可以定量小于10分钟注射后，根据不同的受体小鼠的寄生虫血症。 </li></ol> 3.收集血液样品，并准备流式细胞仪<ol><li>制备50微升的每个样品染色溶液，再加额外，在实验当天如下： <ol><li>除霜6毫JC-1储存在-20℃，在​​二甲基亚砜（DMSO）的等分试样。 </li><li>温暖的50微升地铁每个样品，再加上额外的，到37℃。 </li><li>而连续地涡旋预热地铁，加入JC-1，使最终浓度为12μM。这样做是为了减少JC-1聚集体的形成;如果不聚集形成，不离心管，因为这将防止染色。 注：少量聚集不会影响结果。 </li><li>添加抗CD45 APC eFluor 780和抗CD71 PerCP eFluor 710，以使最终浓度为1微克/毫升。如果执行所述标记的RBC实验中，添加链霉抗PE-Cy7的，以使最终浓度为1微克/毫升。 </li></ol></li><li>通过尾部内的血管的尾部或裂伤的尖端的截肢执行尾出血。将一滴鲜血尾巴上的小重量船或搏命。采血后，确保出血已停止。吸管3微升尾巴血成50微升染色溶液预加热到37℃，并孵育样品在37℃下20分钟。 </li><li>除霜的4mM的赫斯特33342储存在-20℃下在蒸馏水中（如果使用355纳米的激光）或2mM的赫斯特34580储存在-20℃下在蒸馏水中（如果使用405nm的激光）的等分试样。准备500微升每个样品，再加上额外的，对赫斯特的地铁4微米或2微米的解决方案，分别为。 </li><li>加入500μl的4μM赫斯特33342或2微米的Hoechst 34580到样品和在室温下孵育20分钟。 </li><li>离心机细胞在750 XG为3分钟，在4°C，弃上清，加入700μl地铁和流式细胞流动分析。 </li></ol> 4.流式细胞仪<ol><li>用355/488/633 nm激光或405/488/633 nm激光仪器分析样品。 </li><li>通过一个35微米的细胞分析之前，立即滤网过滤器样品。冲洗细胞过滤的样品，并再利用之间蒸馏水。 </li><li>记录足够的事件，以便人口最少包含至少500事件（通常是100 - 10,000,000每个样品总的事件）。 </li><li>激发的Hoechst 33342使用355纳米的激光，并通过一个五十分之四百六十〇滤波器检测事件。激发的Hoechst 34580使用405nm的激光，并通过一个五十分之四百六十〇滤波器检测事件。激发JC-1，抗CD71 PerCP eFluor 710和链霉PE-Cy7的使用488nm的激光，并通过一个四十〇分之五百三检测事件，40分之692和750LP过滤各自LY。激发阿托633和抗CD45 APC eFluor 780使用633纳米激光分别检测通过三十零分之六百七十或750LP过滤事件。 </li><li>使用适当的流式细胞仪分析软件如下进行分析和补偿： <ol><li>选择全细胞和排除噪声，碎屑，并且基于FSC / SSC特性（ 图2A）的血小板。选择基于任一触发脉冲宽度（ 图2B）或通过使用FSC峰面积与高度之比（ 图2C）的单细胞。 </li><li>选择成熟红细胞，并排除红细胞祖细胞和白细胞，基于在PerCP eFluor 710和APC eFluor 780的通道（ 图2D）负荧光。 </li><li>如果执行标记RBC实验，选择基于PE-Cy7的和阿托633荧光标记的红细胞的每个人口和分析这些分开（ 图3A）。 </li><li>基于积极的荧光为JC-1和赫斯特选择pRBCs（ <str翁&gt;图2E-H）。 </li></ol></li></ol> 5.计算和统计<ol><li>除以pRBCs的数目由红细胞的总数目计算原虫（ 即在栅极Q2由事件数在闸门G4划分事件的数量）。当进行两个标记的种群的侵袭测定寄生虫血症可以通过将标记pRBCs的数目由标记的RBCs的数目中，人口（计算即数量的事件，因为他们位于两个栅极L1和Q2，由事件的数量除以在门的L1）。 注意：标记原虫而变化很大，取决于多种因素，例如内源性原虫和循环裂殖子的数量。出于这个原因，它是有帮助的报告原虫比率，其计算公式为经处理的标记的人口由控制标记的人口的寄生虫在每个单独小鼠划分的寄生虫血症。为了纠正可能的染料的影响，报告结果正穿过两个染料组合的平均原虫比例。 </li><li>确定使用一个样本t检验与1为假想的平均比率的统计显着性。 </li></ol>

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作者什么都没有透露。

我们已经描述了两种原虫和裂殖子侵入体内的样品的测定的方法。在寄生虫血症的测量而言，这种方法提供了比以前的方法10-13在于HJ-红细胞的优点可以从pRBCs区分，从而降低假阳性事件的数目。而HJ-红细胞通常在人类中很少见，一些研究报告高水平小鼠15,16使这些细胞和pRBCs为啮齿动物寄生虫血症的精确测量重要的区别。采用这种方法检测原虫的极限是约0.007％14，?...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Life Technologies</td>
			<td>T-3168</td>
			<td>Store small aliquots of 6mM stock solution at -20 &#176;C in DMSO</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-eFluor 780</td>
			<td>eBioscience</td>
			<td>47-0451-80</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD71 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0711-80</td>
			<td>Clone R17217</td>
		</tr>
		<tr>
			<td>Atto 633 NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>1464</td>
			<td>Atto 633-NHS. Store a 2mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>21335</td>
			<td>Biotin-NHS. Store a 25mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>Streptavidin PE-Cy7</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H478</td>
			<td></td>
		</tr>
		<tr>
			<td>35&#181;M filter cap tubes</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD FACSAria II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD Influx</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: CyAn ADP Analyzer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

测量原虫的。 对于寄生虫血症的测定，血细胞应首先被选择，和噪音，碎片和血小板中排除，基于FSC / SSC特性（ 图2A）。根据所使用的流式细胞仪，然后应根据任一触发脉冲宽度（ 图2B），或FSC峰高度以面积比（ 图2C）中选出的单细胞。其余的事件应包括白细胞，染色呈阳性的APC eFluor 780，红细胞祖细胞（包括网织红细胞），染色阳性PerCP ...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

疟原虫侵入和重复内红细胞。裂殖子入侵和原虫的准确评估，因此在评估疟疾感染的过程中是至关重要的。在这里，我们描述了一种流式细胞仪基础的协议用于疟疾的小鼠模型中，这些参数的测量。

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.5K 次观看.  Macquarie University. 疟原虫侵入和重复内红细胞。裂殖子入侵和原虫的准确评估，因此在评估疟疾感染的过程中是至关重要的。在这里，我们描述了一种流式细胞仪基础的协议用于疟疾的小鼠模型中，这些参数的测量。 

视频: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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.

考试流式细胞仪检测胸腺阳性和阴性选择

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

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.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

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

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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.

小鼠与人肺泡巨噬细胞的分离和体外培养

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

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...