Name: fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow
Uploaded: 2016-11-04T14:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow at JoVE.com

We thank Flow Cytometry Laboratory at Virginia-Maryland College of Veterinary Medicine for the use of flow cytometry core facility. This work was supported by XML's startup funds. XL is a Stamps Fellow in the Biomedical and Veterinary Sciences graduate program.

注:MRL / MP-FAS LPR狼疮易感小鼠饲养，并保持在以下机构动物护理和使用委员会（IACUC）在弗吉尼亚理工大学（动物福利保障号码要求的无特定病原体设施（MRL / LPR）: A3208-01）。本研究是在严格按照指南中的建议，美国国立卫生研究院的实验动物的护理和使用进行。所有动物实验均根据IACUC协议＃12-062进行。 1.细胞培养基和排序缓冲区<ol><li>准备完整的细胞培养基（C10），使用RPMI 1640补充有10％胎牛血清，1mM的丙酮酸钠，1％100 MEM非必需氨基酸，10毫米的HEPES，55μM2-巯基乙醇，2mM的L-谷氨酰胺和100U / ml的青霉素 - 链霉素。 </li><li>制备分拣缓冲液（HBSS满），使用1×Hank氏补充有10mM HEPES平衡盐溶液（HBSS），2.5毫克/毫升牛血清白蛋白，0.05毫米氯化镁2和0.2 U / ml的DNA酶I. </li></ol> 2.鼠标解剖<ol><li>准备包含4毫升C10两个6厘米直径的菜肴。将餐具上的冰。 </li><li>安乐死的CO 2吸入鼠标。 </li><li>收获脾肾保持以上ice用C10一个dish， <ol><li>针按下鼠标与夹层板朝上的肚子。通过喷洒70％的乙醇消毒的机构。 </li><li>切开皮肤和皮肤从肌肉壁沿着从耻骨联合到颈部腹侧中线下方分开。 </li><li>切割沿从第一切口到脚踝的后肢皮肤。 </li><li>针在皮肤背面的两侧，并从第一切口的地方膜片切割沿两侧的肌肉壁。 </li><li>针肌壁背面的头部的右侧。 </li><li>定位在小鼠小肠下方和胃旁的右侧脾脏。拿起日的一端Ë脾它从肚子分开。 </li></ol></li><li>切都后肢了包括股骨和胫骨，并尽可能去除所有的肌肉成为可能。 <ol><li>切沿骨盆/髋关节用锋利的剪刀脚踝后肢肌肉，尽量避免血管。 </li><li>通过在骨盆/髋关节和踝/足关节无切削的骨髓内容曝光分开后肢出来。 </li><li>骨头上的清洁剩余肌肉通过用刀片反复刮伤和在接合点切割的肌肉。 </li></ol></li><li>保持骨骼含有冰4毫升C10一个直径6cm培养皿。继续解剖下一个鼠标。 </li></ol> 3.脾细胞分离<ol><li>与反对含4毫升C10，直到没有红条是可见的菜的顶部有70微米的细胞过滤注射器活塞轻轻均质脾脏。 </li><li>通过一个过滤器添加其他6毫升C10入菜第二则总共10个ml的细胞悬液转移到15毫升锥形管中。 </li><li>离心锥形管在800×g离心5分钟，4℃。 </li><li>离心后，吸出上清液和重悬细胞沉淀在2ml 1×红细胞（RBC）溶胞缓冲液。孵育在室温下5分钟。 </li><li>温育后，离心锥形管在800×g离心5分钟，4℃。 </li><li>离心后，吸出上清液和重悬细胞沉淀在1ml C10。放弃任何大的团块（死细胞），并保持管冰上更高版本。 注:如果有许多小团块，用一个70微米的细胞过滤一次过滤细胞悬液。 </li><li>计数使用自动细胞计数器用锥虫蓝的细胞数。 </li></ol> 4.骨髓细胞分离<ol><li>传送用4ml C10骨头研钵中，加入6毫升C10，使在总10毫升C10的体积。 </li><li>通过使用AP轻轻敲碎骨头砂浆estle。 </li><li>用杵释放骨髓成C10轻轻搅拌。 </li><li>通过一个70微米的细胞滤网转移含有骨髓10毫升C10到50毫升锥形管在冰上。加10毫升的新鲜C10成仍含有骨头砂浆。 注:含吸取骨髓上下几次通过过滤网，如果红色团块可见之前的解决方案。 </li><li>重复步骤4.2至4.4倍。最后一次洗涤后，骨头应该显示为白色。 </li><li>离心50ml锥形管中，在800×g离心5分钟，4℃。同时，准备5毫升现成使用密度梯度介质中在RT 15毫升锥形离心管中。 </li><li>离心后，吸出上清液和重悬细胞沉淀在10ml C10。 </li><li>慢慢层上中的5毫升的密度梯度介质的顶部10 ml的细胞悬液中，并保持两相之间的明确的界面。然后离心15毫升锥形管在1363克在30分钟RT（20°C）以设置的加速度为9和减速设置为0（或制动OFF）。 </li><li>离心后，小心地取出顶部8ml上述溶液，并收集在界面（单核细胞的层）2 mL萃取棕黄层到一个新的15毫升锥形管中。 </li><li>基础上加12 1ml冰冷C10到15毫升锥形管含有骨髓单个核细胞，帽和颠倒几次拌匀，然后在800 XG离心管10分钟，4℃。 </li></ol> 5.细胞表面染色用于FACS <ol><li> 样品染色 <ol><li>制备抗小鼠CD16 / 32抗体溶液（1至100稀释于HBSS-充分，5微克/毫升的最终浓度）。 </li><li>在步骤4.10离心后，吸出上清液和重悬细胞沉淀在100μl抗小鼠CD16 / 32的抗体溶液以阻止在单核细胞Fc受体。在冰上孵育10分钟。 </li><li>制备荧光缀合的抗体混合物（蚂蚁的i-小鼠的CD11c-PE的1点40稀释，抗 - 小鼠的CD11b-APC-CY7 1:80稀释，抗 - 小鼠PDCA-1-FITC 1点40的稀释和抗小鼠B220-V500 1点40稀释在HBSS-满于每个样品100μl的最终体积）制造商推荐的。 注意:使用前滴定的抗体是非常重要的。 </li><li> 10分钟温育在步骤5.1.2后，加入4ml冰冷的HBSS-充分和离心机在800×g离心5分钟，4℃以除去未结合的抗小鼠CD16 / 32。 </li><li>离心后，吸出上清液并重新悬浮于100μl荧光缀合的抗体混合物的细胞沉淀。冰上孵育在黑暗中15分钟。 </li><li> 15分钟温育后，加入4ml冰冷的HBSS-充分和离心机在800×g离心5分钟，4℃以除去未结合的荧光标记的抗体。 </li><li>准备FACS装载溶液（500毫微克/毫升的DAPI在HBSS满）。 </li><li>在步骤5.1.6离心后，吸出上清液，重悬在500μ细胞沉淀; l FACS负载的解决方案。细胞悬液转移到12×75mm的圆底管（FACS管），并保持在管上在黑暗中冰上直至1小时内分选。 </li></ol></li><li> 染色赔偿 <ol><li>标签6 FACS管为PE，APC-CY7，FITC，V500，DAPI或不染。以1×10 6个细胞/管进入FACS管，和等分脾细胞用4ml的HBSS-充分洗涤，在800×g离心5分钟，4℃。 注意:使用DNA结合的荧光染料，DAPI，从死细胞区分活细胞。 DAPI可以穿过死细胞的质膜（但不是活细胞），并结合染色体的DNA。 </li><li>吸上清，悬浮细胞沉淀在100微升HBSS满。 </li><li>在每一个相应的FACS管中，加入下列抗体之一:抗鼠CD19-PE 1:40稀释，抗小鼠的CD11b-APC-CY7 1:80稀释，抗小鼠PDCA-1-FITC 1:40稀释或所推荐的厂家专业抗小鼠B220-V500 1:40稀释cturer。离开第 5（DAPI）和第 6 次 （未染色的）FACS管，因为它们。通过摇动拌匀，并在冰上所有的流式细胞仪管在黑暗中15分钟。 </li><li>温育后，在800 xg离心加4ml冰冷的HBSS满到每个管中，离心FACS管5分钟，4℃。 </li><li>离心后，吸出上清液并重新悬浮于500μl冰冷的细胞沉淀冷HBSS满除了在管标记的DAPI，其再悬浮于500μlFACS加载溶液沉淀。保持所有6管冰在黑暗中，直到排序。 </li></ol></li></ol> 6.排序的细胞分选注:上仪和软件排序程序的操作是规范与公司提供的详细的说明。简言之，我们用在20psi 100微米的喷嘴，5之间设置靶细胞浓度-在10万元左右毫升，并调整效率到70％或更高（ 即 ，冲突保持在30％）。 <ol><li>准备用含100 U / ml青霉素，链霉素500微升FBS /管收集流式细胞仪管。 </li><li>采用单染色补偿管从步骤5.2调整细胞分选仪的补偿参数。 <ol><li>记录10,000个细胞中未染色的管设置负门对每个荧光强度。 </li><li>记录10,000个细胞在其他单污点补偿管设置正栅极每个荧光强度。 注意:软件自动计算补偿参数。 </li></ol></li><li>从步骤5.1使用一个样本通过记录3000细胞组分类的细胞群的大门。使用荧光强度为X轴和Y轴曲线图中，栅极靶细胞群体手动作为一组从其他点明显分开的点的参数。 注意:此浇口套是柔性的和任意基于研究人员的要求。如果研究人员期望基于一个或多个标记物纯度较高，将栅极更高根据相应的荧光强度。 </li><li>装入收集管，并开始排序的靶细胞群。 </li><li>保持收集管冰排序，直到所有的样品管完成之后。 </li><li>冰冷的C10添加到收集管达4毫升，帽和反转混合。 </li><li>离心收集管，在800×g离心5分钟，4℃，然后吸出上清液，留下约200微升与细胞沉淀不变。 </li><li>加入1ml冰冷的C10至重悬细胞沉淀，并转移所有细胞悬浮到1.5毫升离心管中。 </li><li>离心机在800×g离心5分钟，4℃1.5 ml管和吸出上清液，留下100微升与细胞沉淀不变。 </li><li>储存在冰上排序的细胞群，直到使用。 </li></ol>

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The authors declare that there is no conflict of interest regarding the publication of this paper.

The protocol described in this manuscript is for high purity enrichment of live pDC that retain the ability to produce IFN&#945;. The applications of this protocol include, but are not limited to, purification of pDC and/or any other mononuclear cells from the bone marrow of MRL/lpr and any other mouse strains for studies of cellular and molecular functions. Several critical steps in this protocol are to ensure high viability and purity of the sorted pDC. The first key step is the release of bone marrow from bones. To minimize damaging bone marrow cells, it is important to gently crack the bones and stir the pestle instead of smashing the bones. The second key step is the enrichment of mononuclear cells with density gradient medium. It is critical to establish a clear interface between the lower phase (density gradient medium) and the upper phase (cell suspension) while loading the cell suspension. The centrifugation condition is also critical for the formation of the buffy coat layer that contains mainly mononuclear cells. After centrifugation, bone debris, red blood cells and high-density neutrophils are excluded from the buffy coat layer and stay at the bottom of the tube. The third key step is incubating the cells with fluorescence-conjugated antibodies at a low temperature (on ice or 4 &#176;C). After the staining, it is also important to wash the cells extensively to ensure the specific antibody binding with minimal false positives or background signals easily detectable by the cell sorter. Finally, the single-stain compensation tubes are important and necessary for the adjustment of compensation parameters to ensure only the correct signals are measured and analyzed on the cell sorter. Besides using isolated splenocytes for the compensation, commercially available compensation beads can be an alternative if cells are limited. In this particular study, as we needed to enrich as many pDC as possible for further functional studies, to avoid bone marrow cell loss, we used splenocytes instead of bone marrow cells for the compensation. Also, it needs to be noted that surface marker like CD11c are expressed on rare populations, so they are not the best choice for compensation. Instead, an alternative marker expressed at a relatively high level on a major population can be used for compensation purpose as long as the antibody is conjugated with the same fluorescence dye. For example, in this study, CD19-PE is used to replace CD11c-PE in the compensation staining of splenocytes. Besides single color controls, fluorescence minus one (FMO) controls are important for beginners, especially to sort a population with markers not apparently separated from other populations. However, in this study, single color controls are adequate as pDC are a distinct population clearly defined and separated from other cells. In addition, we could set the gate slightly smaller than the actual population to improve purity.
In our studies, we only sorted a single population by FACS. In fact, this sorting method can be used broadly not only to enrich several different cell populations simultaneously, but also to isolate any interesting particle targets within a certain size range decided by the cell sorter, as long as they can be labeled with fluorescence. MCS is another cell enrichment method similar to FACS by principle that both are based on antibody detection of cell surface markers. Even though MCS is also widely used, it may not be efficient in various situations. For example, the phenotype of a cell population may change with the microenvironment in vivo or culture condition in vitro. MCS is prone to errors when the cell phenotype changes, as it usually detects only one surface molecule. On the contrary, FACS is based on the combination of several adjustable markers that reflect a more specific and accurate phenotype of a cell population in dynamic conditions. In addition, the gating strategy of FACS gives it a more sophisticated sorting ability, such as sorting Ly6C+ monocytes into Ly6Chigh and Ly6Cmedium subsets, which cannot be done with MCS. 
However, FACS has its limitations as well. As it separates cell populations according to the analysis of fluorescent signals, if the cells or particles of interest have high autofluorescence, it will be difficult to sort the targets. In addition, a designated professional staff is usually required to operate the cell sorter properly. Moreover, the binding of fluorescence-conjugated antibodies to their specific ligands on cells may change the function of sorted cells, affecting the outcomes of subsequent experiments. For example, Siglec-H as a pDC specific surface molecule can be used to sort pDC, but the binding of Siglec-H by its specific antibody has been demonstrated to suppress the ability of pDC to produce IFN&#945;. Therefore, selection of markers for cell sorting needs to be cautious and based on the purpose of the study. 
Furthermore, the high-throughput cell sorter is limited to sorting, with high efficiency, only target cells of certain percentage within the total cell population. In this study, we first used density gradient centrifugation to increase the percentage of pDC through removing red blood cells, high dense granulocytes and debris, which was a better choice than simply adding the red blood cell lysis buffer. If the population is so rare that is less than 1%, the sorting efficiency will drop with significant cell loss. Hence, extremely rare population can be enriched first by other methods, such as negative selection with MCS to remove some of the other cells, and then sorted by FACS to achieve high purity with minimal cell loss. In summary, FACS is an efficient and accurate method to enrich functional pDC from the mouse bone marrow with high purity.

Efficient separation of a cell population of choice from other cells enables studies of the population that may not be possible otherwise. Fluorescence-activated cell sorting (FACS) is a method to enrich an interesting cell population with high purity. 1,2 Different cell types usually express unique molecules, or a unique combination of several molecules, on the plasma membrane that can distinguish one cell population from another. Upon binding of these cell surface molecules by specific fluorescence-conjugated antibodies, a detecting machine called flow cytometer/sorter is able to excite and detect the light signals of different fluorescent dyes that represent different molecule markers on the cells at the single cell level. The combined information consisting of either the presence of a light signal (representing positive expression of the corresponding surface molecule) or the absence of a light signal (representing negative expression of a molecule) defines the phenotype of the cell. After passing through the detector, cells with the same phenotype of interest are diverted towards a designated collecting tube based on electrical charge.
FACS is broadly applied in various studies as long as the population to be enriched is labeled with fluorescence.3-7 It has been used to separate immunoglobulin (Ig)A-coated bacteria from non-IgA coated bacteria in the gut microbiota 8 and sort genetically engineered cell populations expressing fluorescent proteins. 9 Importantly, it has the capacity to separate more than one population simultaneously, which not only saves time and reagents but also allows for more sophisticated study designs. 10 However, FACS also has its limitations. If a population of interest is very rare (less than 1%), the sorting efficiency may be reduced, causing significant cell loss. In addition, some antibody binding may activate intracellular signal transduction that induces functional changes of the sorted cell population. 11 Therefore, the phenotype used for sorting should be selected carefully. 
Other methods exist besides FACS that are also based on cell surface markers for the enrichment of specific cell populations, such as magnetic-activated cell sorting (MCS). 12 Similar to FACS, magnetic beads-conjugated antibodies can target specific cell surface molecules. Upon antibody-antigen interaction, magnetic beads-coated cells can be separated from non-coated cells after passing through a magnetic field. However, only a limited number of molecules can be targeted in MCS, as magnetic beads are, unlike various fluorescent colors in FACS, undistinguishable. It is thus difficult for MCS to define a cell phenotype with a complicated combination of surface markers. 13,14 In addition, MCS is also able to cause unintended activation of target cells.
In our studies of a mouse model of systemic lupus erythematosus (SLE), 15 we intended to purify plasmacytoid dendritic cells (pDC) to investigate their functional changes with disease progression. We first used MCS to enrich pDC from the bone marrow by targeting PDCA-1, a molecule highly and uniquely expressed on murine pDC at steady state. 16 However, the cell purity was unexpectedly low, likely due to the upregulation of PDCA-1 on other cell populations in an inflammatory environment such as SLE.16 Ultimately, we have used FACS with a combination of four surface markers (CD11c, CD11b, B220 and PDCA-1) to separate high-purity pDC as CD11c+CD11b-B220+PDCA-1+ population. Murine pDC has another specific surface marker Siglec-H. We decided not to use Siglec-H, as antibody binding of this molecule represses the function of pDC to produce IFN&#945;. 11

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30396.03</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>1x Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>gibco by life technologies</td><td>14175-079</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec</td><td>130-091-376</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA</td><td>M8266</td><td></td></tr><tr><td>DNase I</td><td>SIGMA</td><td>D4527</td><td></td></tr><tr><td>Red blood cell (RBC) lysis buffer&amp;nbsp;</td><td>eBioscience</td><td>00-4300-54</td><td></td></tr><tr><td>Density gradient medium</td><td>GE Healthcare</td><td>17-1440-02</td><td>Ficoll-Paque Plus&amp;nbsp;</td></tr><tr><td>anti-mouse CD19-PE</td><td>BD Pharmingen</td><td>553786</td><td></td></tr><tr><td>anti-mouse CD11c-PE</td><td>eBioscience</td><td>12-0114-82</td><td></td></tr><tr><td>anti-mouse CD11b-APC-CY7</td><td>BD Pharmingen</td><td>557657</td><td></td></tr><tr><td>anti-mouse PDCA-1-FITC</td><td>eBioscience</td><td>11-3172-81</td><td></td></tr><tr><td>anti-mouse B220-V500&amp;nbsp;</td><td>BD Pharmingen</td><td>561226</td><td></td></tr><tr><td>DAPI</td><td>invitrogen</td><td>D3571</td><td></td></tr><tr><td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td><td>Miltenyi Biotec</td><td>130-092-786</td><td></td></tr><tr><td>BD FACSAria I flow cytometer&amp;nbsp;</td><td>BD Biosciences</td><td>643178</td><td></td></tr><tr><td>BD FACS Diva version 6</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

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.

我们的目的是丰富的骨髓PDC上高纯度，且无其它细胞类型的影响，从年轻和老年学习的PDC对他们产生IFNα能力的功能变化的MRL / lpr狼疮狼疮易感小鼠。使用的第一纯化策略是MCS，其中， 如图1所示，导致只有7.75纯度％的富集。相比于MCS，FACS丰富的pDC，纯度高达96.4％。以确保高纯度，进行一步一步门控策略。正如图2所示，单核细胞进行门控，并通过前向散射（FSC）和侧向散射（SSC）参数从碎片分离。利用FSC-宽度（FSC-W） 与 FSC高度（FSC-H）和SSC-W与SSC-H，单个细胞进行门控，以避免由聚集体产生的假阳性信号。接活的单细胞被门控的DAPI -人口（DAPI +细胞死亡或凋亡）。从活的单细胞，CD11c的+ CD11b的-人口门控，从中B220 + PDCA-1 +人口进一步门控为PDC。排序后的pDC不仅纯度高，而且功能正常。他们不得不产生IFNα 中在CpG的刺激体外 如图3（重新打印与从我们以前的出版物17权限的能力。 <img alt="图1" src="/files/ftp_upload/54641/54641fig1.jpg" /> 图1 的pDC排序的通过任一的MCS和FACS法代表流式细胞示出的pDC的百分比（的CD11c +细胞CD11b - B220 + PDCA-1 +）附图中的纯度。在分选的细胞由MCS（顶部）和FACS（下部），分别。所述的CD11c +细胞CD11b -人口从总活细胞（左），然后的pDC如B220 + PDCA-1选通<html> +细胞的表面CD11c + CD11b的内门-人口（右） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54641/54641fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54641/54641fig2.jpg" /> 图2. 浇注战略pDC的富集FACS按以下顺序的一步一步设门:单核细胞的单细胞FSC，单细胞SSC，以DAPI -活细胞，CD11c的+ CD11b的- ，并B220 + PDCA-1 +作为活的pDC。 pDC的总单体电池是2.95±1.42％;排序的pDC数目是0.88±0.28×10 5个每小鼠用回收率在50％左右。 （12 MRL / lpr狼疮小鼠中，数据被示为平均值的平均值±标准差在95％CI）。<html> 641fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54641/54641fig3.jpg" /> 图3.IFNα生产用流式细胞仪分选的pDC排序骨髓的pDC，用A级的CpG处理（ODN1585; 5μM） 在体外 6小时。在图中，"Y"代表年轻，6周大的老鼠; "O"代表年纪大了，16周大的老鼠。 MRL代表MRL老鼠一样健康对照组; LPR代表MRL / lpr狼疮小鼠为狼疮易感小鼠。 IFNα的培养上清中的浓度用ELISA测定。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001，单向ANOVA。数据显示为平均值±平均值（每组n = 3只小鼠）的标准误差。这个数字是从我们在免疫学杂志出版与转载。17PLOAD / 54641 / 54641fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow at JoVE.com

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

我们使用荧光激活细胞分选来分离浆细胞样树突细胞（PDC）与来自狼疮易感小鼠的骨髓供的pDC的功能研究的高纯度报告的协议。

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

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

fluorescence-activated-cell-sorting-for-purification-plasmacytoid

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  Virginia Polytechnic Institute and State University. 我们使用荧光激活细胞分选来分离浆细胞样树突细胞（PDC）与来自狼疮易感小鼠的骨髓供的pDC的功能研究的高纯度报告的协议。 

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

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.

特定细胞群的净化荧光激活细胞分选（FACS）

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

科研

免疫与感染

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

医学

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

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

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

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

来自常见淋巴祖细胞的浆细胞样树突状细胞的体外生成

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...