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Viral Immunofluorescence with Rift Valley Fever Virus Infected MEFs in a 96 Well Plate
在96孔板中用病毒免疫荧光法检测受裂谷热病毒感染的小鼠胚胎成纤维细胞(MEF)   

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Abstract

Immunofluorescence is a method to detect viral infection in multiple types of host cells. This procedure can be adapted for both high-throughput and low-throughput assays for any virus for which there are antibodies available. Time of infection and virus multiplicity of infection (MOI) vary and should be optimized for each virus and host cell type. Here we give an example of viral immunofluorescence in a 96 well plate with a Rift Valley fever virus (RVFV, strain MP12) infection in mouse embryonic fibroblasts (MEF).

Materials and Reagents

  1. Mouse embryonic fibroblasts (or other host cell)
  2. Rift Valley fever virus MP12 (or other virus)
  3. Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965-084 )
  4. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
  5. Penicillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  6. L-Glutamine (Life Technologies, Gibco®, catalog number: 35050-061 )
  7. Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies, Gibco®, catalog number: 14190-136 )
  8. 0.05% Trypsin (Life Technologies, Gibco®, catalog number: 25300-054 )
  9. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  10. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5368 )
  11. Formaldehyde solution (Sigma-Aldrich, catalog number: 252549 )
  12. TritonX-100 (Sigma-Aldrich, catalog number: 78787 )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2158 )
  14. Primary antibody: Mouse anti-RVFV ID8 (obtained privately from C. Schmaljohn USAMRIID)
  15. Secondary antibody: AlexaFluor goat anti-mouse 488 (Life Technologies, Invitrogen™, catalog number: A11029 )
  16. Hoechst 33342 (Sigma-Aldrich, catalog number: B2261 )
  17. Plate sealing film (Denville Scientific, catalog number: B1212-4 )
  18. Complete DMEM (see Recipes)
  19. Fixative (see Recipes)
  20. PBST (see Recipes)
  21. Block (see Recipes)

Equipment

  1. Black, clear-bottomed 96 well plates (Corning Incorporated, catalog number: 3712 )
  2. T75 flask for cell maintenance (Corning Incorporated, catalog number: 430725 )
  3. Type II Laminar Flow Hood (ESCO Corporation)
  4. Thermo Forma Series II 37 °C Incubator with 5% CO2 (Thermo Fisher Scientific, Forma, catalog number: 3110 )
  5. Hemocytometer (Hausser Scientific Brightline)
  6. Multichannel pipette (Fisherbrand, catalog number: FJ19506 )
  7. Matrix Well Mate (optional) (Thermo Fisher Scientific)
  8. Plate shaker (Barnstead International, catalog number: 4625 )
  9. Centrifuge (Eppendorf, catalog number: 5810R )
  10. Multichannel Manifold Aspirator (Drummond, catalog number: 3-000-096 )
  11. Sterile basins (Thermo Fisher Scientific, catalog number: 13681500 )
  12. Quatracide (Thermo Fisher Scientific, catalog number: 50200423 )
  13. Automated inverted microscope (Molecular Devices Image Express Micro)

Software

  1. Image Analysis software (MetaXpress: Molecular Devices Version 2.0.0.13)

Procedure

  1. Growing cells in 96 well plate
    1. All steps using live cells should be performed in a type II laminar flow hood using sterile technique. MEFs are maintained in T75 flask in complete DMEM at 37 °C with 5% CO2, and passed with 0.05% trypsin 1:10 into a fresh flask every 2-3 days.
    2. Remove spent medium from a confluent flask of MEFs, and wash in 5 ml DPBS. Remove DPBS and treat with 2 ml 0.05% trypsin for a few minutes until the cells dislodge. Add 8 ml of fresh complete DMEM and pipette up and down several times to break up cell clumps and generate a single cell suspension.
    3. Use a hemocytometer to count cells and dilute them in complete DMEM to 200,000 cells/ml in a sterile basin. Use a multichannel pipette to add 100 μl of cells to each well of a 96 well plate (20,000 cells/well).
      Optional: To reduce variability and for high-throughput assays a Well Mate can be used to plate cells. See Filone et al. (2010) for detailed instructions.
    4. Incubate over night at 37 °C/5% CO2 in incubator under standard growth conditions.

  2. Infecting with RVFV
    1. Remove medium from the wells of the 96-well plate using a sterile multi-channel aspirator and vacuum manifold, and replace with 100 μl fresh medium. We sterilize the aspirator in Quatracide for 15 min, then wash in 70% ethanol.
      Notes:
      1. This step may be omitted if a constant final volume is not critical. In particular, we add it when performing drug treatments, so that the concentration of drugs added will be accurate and consistent.
      2. For drug treatments, dilute the drug in DPBS to 20x the final desired concentration and add 5 μl per well. Incubate 1 h at 37 °C prior to infection.
    2. Dilute RVFV stock to desired MOI (generally MOI 1-5) in complete DMEM, and add 10 μl to each well.
      Notes:
      1. Viral infection should be performed at the appropriate biocontainment (RVFV MP12 is BSL2).
      2. Viruses should be stored in small aliquots at -80 °C to avoid repeated freeze-thawing, which can cause the virus to lose titer.
    3. Gently shake the plate for 5 min to ensure even distribution and infection.
    4. Centrifuge plate at room temperature for 1 h at 1,200 RPM.
      Note: This step may be omitted depending on the virus and host cell type, but can greatly enhance the level of infection obtained for some viruses, such as RVFV.
    5. Incubate for 10 h in incubator.

  3. Fixing and staining
    1. Steps are performed at room temperature without shaking unless otherwise noted.
    2. Remove supernatant with aspirator.
    3. Add fixative in 100 μl (4% formaldehyde/PBS) for 10 min.
      Note: Once plate has been fixed, sterile equipment and reagents are no longer needed.
    4. Remove fix using a multi-channel aspirator and vacuum manifold, and add 100 μl PBST (PBS + 0.1% Triton X100) to permeablize the cell membrane. The cells will remain adherent to the bottom of the 96 well plate. Incubate 10 min, remove and repeat PBST wash once more for a total of 2 PBST washes.
    5. Remove liquid and incubate cells in 4 °C block (PBST + 2% BSA) for 10 min. Remove block.
    6. Dilute primary antibody (mouse anti-RVFV ID8 1:1,000) in 4 °C block and add 25 μl to each well using a multi-channel pipette. Seal with a plate sealing film, and incubate over night at 4 °C.
    7. Remove primary antibody, and wash the wells in PBST 3 times for 10 min each.
    8. Dilute fluorescently labeled secondary antibody (goat anti-mouse 488; 1:1,000) and Hoechst 33342 nuclear stain (1:1,000) in block, and add 25 μl to each well.
    9. Incubate plate 1 h at room temperature, protected from light.
    10. Wash plate in PBST 3 times, 10 min each. Leaving 100 μl PBST in each well, seal with a plate sealing film, and store at 4 °C until imaging.

  4. Imaging and analysis
    1. We use an automated inverted microscope at 10x magnification to image multiple sites in each well for both the 4', 6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) channels. The DAPI channel is used to image the Hoechst 33342 nuclear stain, and the FITC channel is used to image the secondary antibody AlexaFluor goat anti-mouse 488, which labels viral antigen.
    2. Image analysis software, such as MetaXpress, is used to measure the total number of cells (DAPI), and infected cells (FITC) for each well. These values are then used to calculate percent infection (FITC/DAPI*100).

Recipes

  1. Complete DMEM
    DMEM
    10% FBS
    100 μg/ml penicillin/streptomycin
    2 mM L-glutamine
    10 mM Hepes
  2. Fixative
    Dilute formaldehyde solution to 4% in PBS
  3. PBST
    Dilute Triton X-100 to 0.1% in PBS
  4. Block
    2% BSA dissolved in PBST
    Stored at 4 °C

Acknowledgments

This protocol is adapted from Filone et al. (2010); Moser et al. (2010); Nakamoto et al. (2012); and Moser et al. (2012).

References

  1. Filone, C. M., Hanna, S. L., Caino, M. C., Bambina, S., Doms, R. W. and Cherry, S. (2010). Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon. PLoS One 5(11): e15483.
  2. Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B. and Cherry, S. (2010). A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics. PLoS Pathog 6(6): e1000954.
  3. Moser, T. S., Sabin, L. R. and Cherry, S. (2010). RNAi screening for host factors involved in Vaccinia virus infection using Drosophila cells. J Vis Exp(42).
  4. Moser, T. S., Schieffer, D. and Cherry, S. (2012). AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog 8(4): e1002661.
  5. Nakamoto, M., Moy, R. H., Xu, J., Bambina, S., Yasunaga, A., Shelly, S. S., Gold, B. and Cherry, S. (2012). Virus recognition by Toll-7 activates antiviral autophagy in Drosophila. Immunity 36(4): 658-667.
  6. Rose, P. P., Hanna, S. L., Spiridigliozzi, A., Wannissorn, N., Beiting, D. P., Ross, S. R., Hardy, R. W., Bambina, S. A., Heise, M. T. and Cherry, S. (2011). Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts. Cell Host Microbe 10(2): 97-104.
  7. Sabin, L. R., Zhou, R., Gruber, J. J., Lukinova, N., Bambina, S., Berman, A., Lau, C. K., Thompson, C. B. and Cherry, S. (2009). Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA virus infection in Drosophila. Cell 138(2): 340-351.
  8. Shelly, S., Lukinova, N., Bambina, S., Berman, A. and Cherry, S. (2009). Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus. Immunity 30(4): 588-598.

简介

免疫荧光是检测多种类型的宿主细胞中的病毒感染的方法。 该方法可适用于任何具有可用抗体的病毒的高通量和低通量测定。 感染时间和病毒感染复数(MOI)不同,应该对每种病毒和宿主细胞类型进行优化。 在这里我们给出在小鼠胚胎成纤维细胞(MEF)中使用Rift Valley热病毒(RVFV,菌株MP12)感染的96孔板中的病毒免疫荧光的实例。

材料和试剂

  1. 小鼠胚胎成纤维细胞(或其他宿主细胞)
  2. MP12(或其他病毒)
  3. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11965-084)
  4. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
  5. 青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140-122)
  6. L-谷氨酰胺(Life Technologies,Gibco ,目录号:35050-061)
  7. Dulbecco's磷酸盐缓冲盐水(DPBS)(Life Technologies,Gibco,目录号:14190-136)
  8. 0.05%胰蛋白酶(Life Technologies,Gibco ,目录号:25300-054)
  9. 乙醇(Sigma-Aldrich,目录号:459844)
  10. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P5368)
  11. 甲醛溶液(Sigma-Aldrich,目录号:252549)
  12. TritonX-100(Sigma-Aldrich,目录号:78787)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2158)
  14. 一级抗体:小鼠抗RVFV ID8(从ATCC Schmaljohn USAMRIID私下获得)
  15. 二抗:AlexaFluor山羊抗小鼠488(Life Technologies,Invitrogen TM,目录号:A11029)
  16. Hoechst 33342(Sigma-Aldrich,目录号:B2261)
  17. 板密封膜(Denville Scientific,目录号:B1212-4)
  18. 完成DMEM(参见配方)
  19. 固定剂(见配方)
  20. PBST(参见配方)
  21. 块(参见配方)

设备

  1. 黑色,透明底96孔板(Corning Incorporated,目录号:3712)
  2. T75烧瓶(Corning Incorporated,目录号:430725)
  3. II型层流罩(ESCO Corporation)
  4. 具有5%CO 2的Thermo Forma系列II 37℃孵育器(Thermo Fisher Scientific,Forma,目录号:3110)
  5. 血细胞计数器(Hausser Scientific Brightline)
  6. 多通道移液管(Fisherbrand,目录号:FJ19506)
  7. Matrix Well Mate(可选)(Thermo Fisher Scientific)
  8. 板振荡器(Barnstead International,目录号:4625)
  9. 离心机(Eppendorf,目录号:5810R)
  10. 多通道集气器吸气器(Drummond,目录号:3-000-096)
  11. 无菌盆(Thermo Fisher Scientific,目录号:13681500)
  12. Quatracide(Thermo Fisher Scientific,目录号:50200423)
  13. 自动倒置显微镜(Molecular Devices Image Express Micro)

软件

  1. 图像分析软件(MetaXpress:Molecular Devices版本2.0.0.13)

程序

  1. 在96孔板中生长细胞
    1. 使用活细胞的所有步骤应当在II型层流罩中使用无菌技术进行。 将MEF在37℃下用5%CO 2保持在完全DMEM中的T75烧瓶中,并且每2-3天用0.05%胰蛋白酶1:10通入新鲜烧瓶中。
    2. 从MEF的汇合瓶中取出用过的培养基,并在5ml DPBS中洗涤。 取出DPBS,用2毫升0.05%胰蛋白酶处理几分钟,直到细胞脱落。 加入8ml新鲜的完全DMEM和 移液器上下数次以破碎细胞团并产生单细胞悬浮液
    3. 使用血细胞计数器细胞,并将其在完全DMEM中稀释至200,000个细胞/ml,在无菌盆中。 使用多通道移液器添加100微升的细胞到96孔板的每个孔(20,000个细胞/孔)。
      可选:为了降低变异性和进行高通量测定,可以使用Well Mate平板细胞。 参见Filone et al。 (2010),了解详细说明。
    4. 在标准生长条件下,在37℃/5%CO 2孵育箱中孵育过夜。

  2. 用RVFV感染
    1. 使用无菌多通道吸气器和真空歧管从96孔板的孔中删除培养基,并更换为100μl新鲜培养基。 我们在Quatracide中对吸气器消毒15分钟,然后在70%乙醇中洗涤 注意:
      1. 如果恒定的最终体积不是关键的,则可以省略该步骤。 特别是,我们在进行药物治疗时添加它,以使添加的药物浓度准确和一致。
      2. 对于药物治疗,将DPBS中的药物稀释至最终所需浓度的20倍,并每孔加入5μl。 在感染前在37℃孵育1小时。
    2. 在完全DMEM中稀释RVFV原液至所需MOI(通常为MOI 1-5),并向每个孔中加入10μl。
      注意:
      1. 病毒感染应在适当的生物防范(RVFV MP12为BSL2)下进行。
      2. 病毒应该以小等分试样储存在-80℃,以避免反复冻融,
    3. 轻轻摇动平板5分钟,以确保均匀分布和感染
    4. 将板在室温下以1,200RPM离心1小时 注意:根据病毒和宿主细胞类型,此步骤可能会被省略,但可以大大提高某些病毒(如RVFV)获得的感染水平。
    5. 在孵化器中孵育10小时。

  3. 固定和染色
    1. 步骤在室温下进行,除非另有说明,否则摇动
    2. 用吸气器除去上清液。
    3. 在100μl(4%甲醛/PBS)中加入固定剂10分钟 注意:一旦固定板,就不再需要无菌的设备和试剂。
    4. 使用多通道吸气器和真空歧管去除修复,并添加100微升PBST(PBS + 0.1%Triton X100)渗透细胞膜。 细胞将保持粘附到96孔板的底部。 孵育10分钟,取出并重复PBST洗涤一次,共2次PBST洗涤
    5. 取出液体,孵育细胞在4℃块(PBST + 2%BSA)10分钟。 删除块。
    6. 稀释初级抗体(小鼠抗RVFV ID8 1:1,000)在4℃块,并添加25微升到每个孔使用多通道移液器。 用板密封膜密封,并在4℃下孵育过夜
    7. 取出一抗,并在PBST中洗涤孔3次,每次10分钟
    8. 稀释荧光标记的第二抗体(山羊抗小鼠488; 1:1,000)和Hoechst 33342核染色(1:1,000)块,并添加25微升到每个孔。
    9. 在室温下孵育平板1小时,避光保护
    10. 将板在PBST中洗涤3次,每次10分钟。 在每个孔中留下100μlPBST,用板密封膜密封,并在4℃下储存直到成像

  4. 成像和分析
    1. 我们使用自动倒置显微镜在10倍放大图像为4',6-二脒基-2-苯基吲哚(DAPI)和异硫氰酸荧光素(FITC)渠道的每个孔中的多个网站。 DAPI通道用于成像Hoechst 33342核染色,FITC通道用于成像二抗AlexaFluor山羊抗小鼠488,其标记病毒抗原。
    2. 使用图像分析软件(例如MetaXpress)来测量每个孔的细胞总数(DAPI)和感染细胞(FITC)。 这些值然后用于计算感染百分比(FITC/DAPI * 100)。

食谱

  1. 完成DMEM
    DMEM
    10%FBS
    100μg/ml青霉素/链霉素 2mM L-谷氨酰胺 10 mM Hepes
  2. 固定剂
    将甲醛溶液稀释至4%的PBS溶液
  3. PBST
    将Triton X-100稀释至0.1%的PBS溶液
  4. 阻止
    2%BSA溶于PBST中 储存在4°C

致谢

该协议改编自Filone等人(2010); Moser等人(2010); Nakamoto (2012); 和Moser等人(2012)。

参考文献

  1. Filone,C.M.,Hanna,S.L.,Caino,M.C.,Bambina,S.,Doms,R.W.and Cherry,S。(2010)。 对人类细胞和昆虫宿主的 裂谷热病毒感染是由 蛋白激酶Cε。 PLoS One 5(11):e15483。
  2. Moser,T.S.,Jones,R.G.,Thompson,C.B.,Coyne,C.B.and Cherry,S。(2010)。 一个kinome RNAi屏幕通过控制将AMPK标识为促进痘病毒进入 肌动蛋白动力学。 PLoS Pathog 6(6):e1000954。
  3. Moser,T.S.,Sabin,L.R。和Cherry,S。(2010)。 RNAi筛选参与牛痘病毒感染的宿主因子 果蝇细胞。 J Vis Exp (42)。
  4. Moser,T.S.,Schieffer,D。和Cherry,S。(2012)。 AMP激活的激酶通过抑制脂肪酸来限制裂谷热病毒感染 PLoS Pathog 8(4):e1002661。
  5. Nakamoto,M.,Moy,R. H.,Xu,J.,Bambina,S.,Yasunaga,A.,Shelly,S.S.,Gold,B。和Cherry,S。 Toll-7的病毒识别在果蝇中激活抗病毒自噬。 Immunity 36(4):658-667。
  6. Rose,P.P.,Hanna,S.L.,Spiridigliozzi,A.,Wannissorn,N.,Beiting,D.P.,Ross,S.R.,Hardy,R.W.,Bambina,S.A.,Heise,M.T.and Cherry, 天然抗性相关巨噬细胞蛋白是辛德毕斯病毒的细胞受体。昆虫和哺乳动物宿主。 Cell Host Microbe 10(2):97-104。
  7. Sabin,L.R.,Zhou,R.,Gruber,J.J.,Lukinova,N.,Bambina,S.,Berman,A.,Lau,C.K.,Thompson,C.B.and Cherry,S。 Ars2调节miRNA和siRNA依赖性沉默并抑制果蝇中的RNA病毒感染。 138(2):340-351
  8. Shelly,S.,Lukinova,N.,Bambina,S.,Berman,A。和Cherry,S。(2009)。 自噬是果蝇免疫反应水泡性口炎的重要组成部分 病毒。 免疫力 30(4):588-598
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Copyright: © 2012 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. Moser, T. S. and Cherry, S. (2012). Viral Immunofluorescence with Rift Valley Fever Virus Infected MEFs in a 96 Well Plate. Bio-protocol 2(23): e297. DOI: 10.21769/BioProtoc.297.
  2. Moser, T. S., Schieffer, D. and Cherry, S. (2012). AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog 8(4): e1002661.
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