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Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells
对疟原虫感染的红细胞进行示踪染色以观察免疫吞噬效应   

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Abstract

Efficient staining methods to identify Plasmodium-infected red blood cells (iRBCs) are crucial to discriminate precisely the immune cells responsible for their elimination from circulation. Here, we describe the protocol for the purification of iRBCs and their subsequent staining with the vital dyes Cell Tracer Violet (CTV) or CellTracker Red CMTPX (CMTPX), both of which readily diffuse into cells and bind covalently to intracellular amines. The iRBCs stained by using this protocol were used in ex vivo phagocytosis assays, to determine the ability of splenic dendritic cells of phagocytizing these parasites (Borges da Silva et al., 2015).

Materials and Reagents

  1. 15 ml tubes (TPP, catalog number: 91015 )
  2. 1 ml syringes (BD Biosciences, catalog number: 309659 )
  3. B6.Rag2-/- (RAGKO) mice (Jackson Laboratories, C57BL/6)
  4. Plasmodium chabaudi (AS strain)
  5. RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
  6. Fetal bovine serum heat inactivated (Thermo Fisher Scientific, catalog number: 10437028 )
  7. Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
  8. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
  9. Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070 )
  10. 2-mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023 )
  11. Cell Tracer Violet (CTV) (Thermo Fisher Scientific, catalog number: C34557 )
  12. CellTracker Red CMTPX (CMTPX) (Thermo Fisher Scientific, catalog number: C34552 )
  13. Halothane (Sigma-Aldrich, catalog number: H0150000 )
  14. NaCl
  15. KCl
  16. Na2HPO4
  17. KH2PO4
  18. Percoll (GE Healthcare Dharmacon, catalog number: 17089101 )
  19. 1x phosphate buffered saline (PBS) (see Recipes)
  20. 74% Percoll (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5804 )
  2. 1-ml micropipettes
  3. 25 gauge needle (BD Biosciences, catalog number: 305122 )
  4. Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
  5. Epi-Fluorescent microscope (Leica Microsystems, model: DM6000B )

Procedure

  1. 6-8 weeks old RAG2KO mice (used here because we observed higher proportion of mature parasite forms in circulation instead bound to endothelial cells-personal observation) are infected i.p. with 106 Pc-infected iRBCs. The infection in RAGKO allows the posterior collection of a greater proportion of mature iRBCs (Borges da Silva, personal observation).
  2. Seven days after infection, mice are euthanized with halothane by inhalation and bled by cardiac puncture with a 1 ml syringe with a 25 gauge needle, in the presence of approximately 50 µl of heparin. A volume of approximately 500 µl of blood is enough to collect up to 5 x 108 mature iRBCs.
    Note: Verify the institution approved ethical regulations on animal welfare if other euthanization methods are in place.
  3. The blood collected is diluted in approximately 1 ml of PBS 1x (1:1), and placed on a 74% Percoll solution in 15 ml tubes (5 ml). The tubes are centrifuged for 30 min at 2,500 x g, at room temperature with acceleration/break of 5/0, respectively.
  4. After the centrifugation, the mature iRBCs are located as a layer above the 74% Percoll (Figure 1), and are collected by using 1-ml micropipettes into new 15 ml tubes. The mature iRBCs are then washed twice with 1x PBS.


    Figure 1. Percoll-based purification of mature iRBCs. A. A representative picture of 15-ml conical tubes containing RBCs over Percoll 74% before centrifugation. B. A representative picture of 15-ml conical tubes containing the layer with mature iRBCs after Percoll 74% centrifugation.

  5. After washing, a sample of the mature iRBCs (approximately 5 µl) is collected for a blood smear, to confirm the purity for mature iRBCs versus non-mature iRBCs (Figure 2) by counting the percentage of mature iRBCs versus non-mature iRBCs in the blood smear (at this point, it is expected up to 97% of mature iRBCs in the blood smear). Then, 1 x 108 mature iRBCs (resuspended in 2 ml of PBS) are incubated with 5 µM of CTV or with 5 µM of CMTPX (both dyes previously diluted in DMSO and with final suspension volume of 2 ml), at 37 °C, for 30 min.
    Notes:
    1. The expected purity for the samples is between 90-99% of mature iRBCs.
    2. During the incubation, mix the cells gently for homogenization.


    Figure 2. Mature iRBCs purification assessment. A. A representative picture of a blood smear of a Plasmodium-infected mouse, pre-Percoll enrichment. B. A representative picture of a blood smear of post-Percoll enrichment blood of the same mice. Obs: The infected RBCs can be detected by the bright green spots (these images were acquired in Bright Field, in a Leica DM6000B Epi-Fluorescent Microscope).

  6. After incubation, the mature iRBCs are washed twice with 1x PBS for removal of excess dye. The stained mature iRBCs are, then, ready for use in phagocytosis assays as described in (Borges da Silva et al., 2015).

Notes

  1. This procedure usually yields between 95-97% of mature iRBCs purity. However, when done with mice with lower parasitemia levels, there is a drop in these numbers up to 50% purity. Thus, it is preferential to collect blood from RAG2KO mice with > 50% parasitemia (verified upon a blood smear previously done to select mice).

Recipes

  1. 1x phosphate buffered saline (PBS)
    Dissolve the following in 800 ml distilled H2O
    8 g NaCl
    0.2 g KCl
    1.44g Na2HPO4
    0.24 g KH2PO4
    Adjust pH to 7.4
    Adjust volume to 1 L with additional distilled H2O
    Sterilize the solution
    Note: Adjust the pH using HCl and NaOH.
  2. 74% Percoll
    Percoll is commercially available at a 10x concentration. To make a 1x concentrated Percoll solution (which is equivalent to a 100% Percoll solution), it is necessary to dilute 9 parts of Percoll (9 ml GE Healthcare) with 1 part of 10x PBS (1 ml).
    To make 74% Percoll, it is necessary to dilute 3.7 ml of 100% Percoll with 1.3 ml of 1x PBS.

Acknowledgments

HBdS was supported by an award from FAPESP (number: 2014/008105) and MRDL was supported by a grant from FAPESP (number: 2013/071402), and from CNPq 303676/20140 (MRDL) and 448765/20144 (MRDL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.

简介

Efficient staining methods to identify Plasmodium-infected red blood cells (iRBCs) are crucial to discriminate precisely the immune cells responsible for their elimination from circulation. Here, we describe the protocol for the purification of iRBCs and their subsequent staining with the vital dyes Cell Tracer Violet (CTV) or CellTracker Red CMTPX (CMTPX), both of which readily diffuse into cells and bind covalently to intracellular amines. The iRBCs stained by using this protocol were used in ex vivo phagocytosis assays, to determine the ability of splenic dendritic cells of phagocytizing these parasites (Borges da Silva et al., 2015).

材料和试剂

  1. 15ml管(TPP,目录号:91015)
  2. 1ml注射器(BD Biosciences,目录号:309659)
  3. B6.Rag2 -/- (RAGKO)小鼠(Jackson Laboratories,C57BL/6)
  4. 疟原虫(AS株)
  5. RPMI 1640(Thermo Fisher Scientific,目录号:11875093)
  6. 胎牛血清热灭活(Thermo Fisher Scientific,目录号:10437028)
  7. 青霉素 - 链霉素(Thermo Fisher Scientific,目录号:15140122)
  8. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
  9. 丙酮酸钠(Thermo Fisher Scientific,目录号:11360070)
  10. 2-巯基乙醇(Thermo Fisher Scientific,目录号:21985023)
  11. Cell Tracer Violet(CTV)(Thermo Fisher Scientific,目录号:C34557)
  12. CellTracker Red CMTPX(CMTPX)(Thermo Fisher Scientific,目录号:C34552)
  13. 氟烷(Sigma-Aldrich,目录号:H0150000)
  14. NaCl
  15. KCl
  16. Na HPO 4
  17. KH 2 PO 4
  18. Percoll(GE Healthcare Dharmacon,目录号:17089101)
  19. 1×磷酸盐缓冲盐水(PBS)(见Recipes)
  20. 74%Percoll(见配方)

设备

  1. 离心机(Eppendorf,型号:5804)
  2. 1 ml微量移液器
  3. 25号针(BD Biosciences,目录号:305122)
  4. 层流罩(AirClean Systems,目录号:AC8000HLF)
  5. Epi荧光显微镜(Leica Microsystems,型号:DM6000B)

程序

  1. 6-8周龄RAG2KO小鼠(此处使用,因为我们观察到更高比例的循环中的成熟寄生虫形式,而不是结合内皮细胞 - 个体观察)用10 6 - - 感染的iRBC。 RAGKO的感染允许后部收集更大比例的成熟iRBC(Borges da Silva,个人观察)。
  2. 感染后7天,通过吸入用氟烷安乐死小鼠,在存在约50μl肝素的情况下用具有25号针的1ml注射器通过心脏穿刺放血。大约500μl体积的血液足以收集高达5×10 8个成熟iRBC。
    注意:如果其他安乐死方法到位,请验证机构批准的动物福利道德规范。
  3. 收集的血液在约1ml PBS 1x(1:1)中稀释,并置于15ml管(5ml)中的74%percoll溶液中。将试管在室温下以2,500×g离心30分钟,加速/断裂为5/0。
  4. 离心后,成熟iRBC位于74%percoll上方的层(图1),并通过使用1ml微量移液器收集到新的15ml管中。然后将成熟的iRBC用1x PBS洗涤两次

    图1.成熟iRBC的基于Percoll的纯化 A.在离心之前,含有RBC的15-ml锥形管在Percoll 74%上的代表性图片。 B.在Percoll 74%离心后含有具有成熟iRBC的层的15-ml锥形管的代表性图片。

  5. 洗涤后,收集成熟iRBC样品(约5μl)用于血液涂片,以通过计数成熟iRBC与非成熟iRBC的百分比来确认成熟iRBC与非成熟iRBC的纯度(图2)血液涂片(在这一点上,预期高达97%的成熟iRBC在血液涂片中)。然后,将1×10 8个成熟iRBC(重悬于2ml PBS中)与5μM的CTV或5μM的CMTPX(两种染料都预先在DMSO上稀释,最终悬浮液体积为2 ml),37℃,30分钟 注意:
    1. 样品的预期纯度在成熟iRBC的90-99%之间。
    2. 在孵育过程中,轻轻混合细胞以进行匀浆。


    图2.成熟的iRBCs纯化评估。A.疟原虫感染的小鼠血液涂片的代表性图片,Pre-Percoll富集。 B.相同小鼠的后Percoll富集血液的血涂片的代表性图片。 Obs:感染的RBC可以通过亮绿色斑点检测(这些图像在Bright Field中,在Leica DM6000B Epi荧光显微镜中获取)。

  6. 孵育后,将成熟iRBC用1x PBS洗涤两次以除去过量的染料。然后,染色的成熟iRBC准备用于如(Borges da Silva等人,2015)中所述的吞噬作用测定。

笔记

  1. 该程序通常产生95-97%的成熟iRBCs纯度。然而,当用具有较低寄生虫血症水平的小鼠进行时,这些数字的下降达到50%的纯度。因此,优选从RAG2KO小鼠中收集血液, 50%寄生虫血症(通过先前对选择小鼠进行的血涂片验证)。

食谱

  1. 1×磷酸盐缓冲盐水(PBS)
    将以下物质溶于800ml蒸馏H 2 O中 8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4。 0.24g KH 2 PO 4
    将pH调节至7.4
    用额外的蒸馏H 2 O 2调节体积至1L 消毒溶液
    注意:使用HCl和NaOH调节pH。
  2. 74%Percoll
    Percoll可以10x浓度商购获得。为了制备1x浓缩Percoll溶液(相当于100%Percoll溶液),需要用1份10x PBS(1ml)稀释9份Percoll(9ml GE Healthcare)。
    为了制备74%的Percoll,需要用1.3ml的1x PBS稀释3.7ml的100%Percoll。

致谢

HBDS获得FAPESP(编号:2014/008105)的奖励支持,MRDL获得FAPESP(编号:2013/071402),CNPq 303676/20140(MRDL)和448765/20144(MRDL)的资助。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。

参考文献

  1. Borges da Silva,H.,Fonseca,R.,Cassado Ados,A.,Machado de Salles,E.,de Menezes,MN,Langhorne,J.,Perez,KR,Cuccovia,IM,Ryffel,B.,Barreto, VM,Marinho,CR,Boscardin,SB,Alvarez,JM,D'Imperio-Lima,MR和Tadokoro,CE(2015)。  体内方法揭示了DC在CD 4 + T细胞激活和寄生物清除中的关键作用在实验性血液阶段疟疾的急性期期间。 PLoS Pathog 11(2):e1004598。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Silva, H. B., Tadokoro, C. E. and D’Império-Lima, M. R. (2016). Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells. Bio-protocol 6(15): e1884. DOI: 10.21769/BioProtoc.1884.
  2. Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
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