Protocol for improved resolution of plasma cell subpopulations by flow cytometry.
Protocol for improved resolution of plasma cell subpopulations by flow cytometry.
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DOI：
10.1002/eji.201746944
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发表时间：
2017-08
影响因子：
5.4
通讯作者：
Allman D
中科院分区：
文献类型：
--
作者：
Wilmore JR;Jones DD;Allman D
分泌抗体的浆细胞在保护性免疫和抗体介导的自身免疫性疾病中起关键作用。在免疫应答期间，一小部分新生成的浆细胞进入骨髓（BM）或小肠固有层（siLP），在那里它们似乎无限期存活[1 - 3]，从而长时间维持抗体滴度。影响浆细胞生成和存活的因素仍然是个谜。事实上，最近的数据表明，小鼠和人的BM也含有相当数量的短寿命细胞[4，5]，这就提出了关于未成熟浆细胞在进入BM后如何实现长寿的问题。其他最近的工作也揭示了浆细胞可以分泌免疫调节细胞因子[6，7]，增加了更好地定义具有这些新功能的浆细胞的动机。然而，常规表征浆细胞亚群的能力受到了阻碍，因为它们在复杂的免疫组织如脾、siLP和BM中相对缺乏，加上缺乏标准化的流式细胞术和相关方法来鉴定这些细胞，也许用于鉴定小鼠浆细胞的最可靠的表面标志物是CD 138，也称为Syndecan-1。然而，应该注意的是，CD 138也被BM中的许多B细胞前体表达，尽管表面密度较低[8]。从前B细胞和其他细胞类型中分离浆细胞的一个主要进展是产生了浆细胞必需的转录因子Blimp1的报告小鼠。已经产生了两条这样的线。Nutt及其同事将编码GFP的cDNA插入Blimp1基因座的3-prime UTR [9]，导致C57BL/6回交B6。Blimp1 +/GFP小鼠。或者，Meffre和同事们产生了一种细菌人工染色体，其中YFP表达由Blimp1基因座控制[10]。两种策略似乎都忠实地标记Blimp1阳性细胞。然而，因为它并不总是可行的或实际的繁殖与Blimp1报告小鼠的目的小鼠，我们试图建立一个流式细胞仪解决标准近交系小鼠缺乏Blimp1报告的未成熟和成熟的浆细胞亚群的协议。我们的方法可以应用于任何组织，只要可以产生活细胞的单细胞悬浮液，但在BM和脾制备物中观察到最佳结果。这种方法的一个关键的额外表面抗原是干细胞抗原-1（Sca-1），也称为Ly6A/E。
Antibody-secreting plasma cells play critical roles in protective immunity and antibody mediated autoimmune disease. During immune responses a small fraction of newly generated plasma cells enter either the bone marrow (BM) or the lamina propria of the small intestine (siLP) where they appear to survive indefinitely [1-3], thus maintaining antibody titers for extended periods. The factors that influence the generation and survival of plasma cells remain mysterious. Indeed, recent data suggest that the BM in mice and people also harbors considerable numbers of short-lived cells [4, 5], raising questions about how immature plasma cells achieve longevity after they enter the BM. Other recent work has also revealed that plasma cells can secrete immunoregulatory cytokines [6, 7], adding to the motivation to better define plasma cells with these novel functions. Yet the ability to characterize plasma cell subpopulations routinely has been hampered by their relatively paucity in complex immune tissues such as the spleen, siLP, and BM, coupled with the lack of standardized flow cytometric and related methods to identify such cells.Perhaps the most reliable surface marker for identifying plasma cells in mice is CD138, also known as Syndecan-1. However it should be noted that CD138 is also expressed, albeit at lower surface densities, by many B cell precursors in the BM [8]. A major advance for resolving plasma cells from pre-B cells and other cell types was the generation of reporter mice for the plasma cell-requisite transcription factor Blimp1. Two such lines have been generated. Nutt and colleagues inserted a cDNA encoding GFP into the 3-prime UTR of the Blimp1 locus [9], resulting in C57BL/6 backcrossed B6. Blimp1+/GFP mice. Alternatively Meffre and colleagues generated a bacterial artificial chromosome in which YFP expression is controlled by the Blimp1 locus [10]. Both strategies appear to faithfully mark Blimp1-positive cells. However, because it is not always feasible or practical to breed mice of interest with Blimp1 reporter mice, we sought to establish a flow cytometric protocol for resolving immature and mature plasma cell subsets in standard inbred mice lacking a Blimp1 reporter. Our approach can be applied to any tissue provided that single cell suspensions of viable cells can be generated, but optimal results are seen in BM and spleen preparations. A key additional surface antigen for this approach is Stem cell antigen-1 (Sca-1), also known as Ly6A/E.
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影响因子：
32.4
作者：
Rutishauser, Rachel L.;Martins, Gislaine A.;Kalachikov, Sergey;Chandele, Anmol;Parish, Ian A.;Meffre, Eric;Jacob, Joshy;Calame, Kathryn;Kaech, Susan M.
通讯作者：
Kaech, Susan M.
影响因子：
64.8
作者：
Fritz, Joerg H.;Rojas, Olga Lucia;Simard, Nathalie;McCarthy, Douglas D.;Hapfelmeier, Siegfried;Rubino, Stephen;Robertson, Susan J.;Larijani, Mani;Gosselin, Jean;Ivanov, Ivaylo I.;Martin, Alberto;Casellas, Rafael;Philpott, Dana J.;Girardin, Stephen E.;McCoy, Kathy D.;Macpherson, Andrew J.;Paige, Christopher J.;Gommerman, Jennifer L.
通讯作者：
Gommerman, Jennifer L.
影响因子：
32.4
作者：
Halliley JL;Tipton CM;Liesveld J;Rosenberg AF;Darce J;Gregoretti IV;Popova L;Kaminiski D;Fucile CF;Albizua I;Kyu S;Chiang KY;Bradley KT;Burack R;Slifka M;Hammarlund E;Wu H;Zhao L;Walsh EE;Falsey AR;Randall TD;Cheung WC;Sanz I;Lee FE
通讯作者：
Lee FE
DOI：
10.1084/jem.20040973
发表时间：
2004-10-18
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Kallies A;Hasbold J;Tarlinton DM;Dietrich W;Corcoran LM;Hodgkin PD;Nutt SL
通讯作者：
Nutt SL
影响因子：
64.8
作者：
通讯作者：
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