荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

摘要

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。

摘要

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.

引言

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 IFNα. ¹¹

研究方案

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

1. 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。
2. 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.

2.鼠标解剖

1. 准备包含4毫升C10两个6厘米直径的菜肴。将餐具上的冰。
2. 安乐死的CO ₂吸入鼠标。
3. 收获脾肾保持以上ice用C10一个dish，
   1. 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。
   2. 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。
   3. 切割沿从第一切口到脚踝的后肢皮肤。
   4. 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。
   5. 针肌壁背面的头部的右侧。
   6. 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。
4. 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。
   1. 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。
   2. 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。
   3. 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。
5. 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。

3.脾细胞分离

1. 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。
2. 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。
3. 离心锥形管在800×g离心5分钟，4℃。
4. 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。
5. 温育后，离心锥形管在800×g离心5分钟，4℃。
6. 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。
7. 计数使用自动细胞计数器用锥虫蓝的细胞数。

4.骨髓细胞分离

1. 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。
2. 通过使用AP轻轻敲碎骨头砂浆estle。
3. 用杵释放骨髓成C10轻轻搅拌。
4. 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。
5. 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。
6. 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。
7. 离心后，吸出上清液和重悬细胞沉淀在10ml C10。
8. 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）。
9. 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。
10. 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℃。

5.细胞表面染色用于FACS

1. 样品染色
   1. 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）。
   2. 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。
   3. 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。
   4. 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。
   5. 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。
   6. 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。
   7. 准备FACS装载溶液（500毫微克/毫升的DAPI在HBSS满）。
   8. 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。
2. 染色赔偿
   1. 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。
   2. 吸上清，悬浮细胞沉淀在100微升HBSS满。
   3. 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。
   4. 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℃。
   5. 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。

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％）。

1. 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。
2. 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。
   1. 记录10,000个细胞中未染色的管设置负门对每个荧光强度。
   2. 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。
3. 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。
4. 装入收集管，并开始排序的靶细胞群。
5. 保持收集管冰排序，直到所有的样品管完成之后。
6. 冰冷的C10添加到收集管达4毫升，帽和反转混合。
7. 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。
8. 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。
9. 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。
10. 储存在冰上排序的细胞群，直到使用。

结果

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?...

讨论

The protocol described in this manuscript is for high purity enrichment of live pDC that retain the ability to produce IFNα. 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 mi...

材料

Name	Company	Catalog Number	Comments
RPMI 1640	gibco by life technologies	11875-093
Fetal bovine serum	HyClone	SH30396.03
Sodium pyruvate	gibco by life technologies	11360-070
MEM non-essential amino acids	gibco by life technologies	11140-050
HEPES	gibco by life technologies	15630-080
2-mercaptoethanol	gibco by life technologies	21985-023
L-glutamine	gibco by life technologies	25030-164
Penicillin-Streptomycin	gibco by life technologies	15140-122
1x Hank’s Balanced Salt Solution	gibco by life technologies	14175-079
MACS BSA Stock Solution	Miltenyi Biotec	130-091-376
MgCl2	SIGMA	M8266
DNase I	SIGMA	D4527
Red blood cell (RBC) lysis buffer	eBioscience	00-4300-54
Density gradient medium	GE Healthcare	17-1440-02	Ficoll-Paque Plus
anti-mouse CD19-PE	BD Pharmingen	553786
anti-mouse CD11c-PE	eBioscience	12-0114-82
anti-mouse CD11b-APC-CY7	BD Pharmingen	557657
anti-mouse PDCA-1-FITC	eBioscience	11-3172-81
anti-mouse B220-V500	BD Pharmingen	561226
DAPI	invitrogen	D3571
Plasmacytoid Dendritic Cell Isolation Kit II, mouse	Miltenyi Biotec	130-092-786
BD FACSAria I flow cytometer	BD Biosciences	643178
BD FACS Diva version 6	BD Biosciences