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Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells
肝星状细胞中外泌体的分离、BODIPY标记和摄取   

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Journal of Virology
Mar 2017

Abstract

Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes by hepatic stellate cells.

Keywords: Exosomes (外泌体), Hepatic stellate cells (肝星状细胞), BODIPY (BODIPY), Dynamic light scattering (动态光散射), Hepatocytes (肝细胞), Hepatitis C virus (丙型肝炎病毒)

Background

Exosomes are small membrane-bound extracellular vesicles of 40-150 nm in diameter. They have been identified in all body fluids, including urine, amniotic fluid, serum, saliva, breast milk, cerebrospinal fluid, nasal secretions and in the supernatant of tissue cultured cell lines. Exosomes mediate cell to cell communication through transfer of microRNAs (miRs), mRNAs, and proteins, etc. which can be taken up by neighboring or distant cells and subsequently promote signaling in recipient cells (Schorey et al., 2015). Exosomes are enriched in proteins, including members of the tetraspanin family (CD9, CD81, CD63), heat shock proteins (Hsp60, Hsp70, Hsp90) and proteins of the multivesicular bodies (annexins, RabGTPases and endosomal sorting complexes required for transport [ESCRT] proteins). Recent studies suggest that exosomes can influence immune response (Li et al., 2013; Sun et al., 2016), facilitate productive infection in naïve hepatoma cells (Ramakrishnaiah et al., 2013; Shrivastava et al., 2015), disease progression (Devhare et al., 2017) as well as drug resistance to anti-cancer therapy (Qu et al., 2016). Most commonly used techniques for isolation of exosomes involves ultracentrifugation (UC) and ExoQuick (EQ) precipitation. In hepatitis C virus (HCV) infection, crosstalk between liver resident cells including hepatocytes, macrophages, endothelial cells, lymphocytes and stellate cells play an important role in disease progression. We have shown earlier that HCV infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in the cell culture supernatants (Shrivastava et al., 2015). These exosomes carried miR-19a that induces fibrogenesis in hepatic stellate cells that might lead to liver fibrosis during HCV infection (Devhare et al., 2017). Here, we provide a protocol for labeling of exosomes with green fluorescent lipophilic dye, BODIPY that enables real-time monitoring and localization of exosomes within the recipient hepatic stellate cells.

Materials and Reagents

  1. Serological pipettes (5 ml, 10 ml, 25 ml) (Corning)
  2. 50 ml polypropylene centrifuge tubes (Corning, catalog number: 430829 )
  3. T-25 cm2 cell culture flask (Corning, catalog number: 430639 )
  4. 100 mm tissue culture treated culture dish (Corning, catalog number: 430167 )
  5. Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm for SW41Ti rotor (Beckman Coulter, catalog number: 344059 )
  6. 15 ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
  7. 4-well chambered slides (Thermo Fisher Scientific, Nunc, catalog number: 177437 )
  8. Aluminium foil (Fisher Scientific, FisherbrandTM, catalog number: 01-213-102 )
  9. 0.2 μm size filter (Acrodisc Syringe Filters) (Pall, catalog number: 4612 )
  10. Huh7.5 cell line (Human hepatocellular carcinoma cell line)
  11. Rep2a-Rluc cell line: Huh7.5 cells harboring genome-length replicon of hepatitis C virus
  12. LX2 cells (immortalized human hepatic stellate cells)
  13. DMEM medium (Sigma-Aldrich, catalog number: D5796 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  15. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P0781 )
  16. Phosphate buffered saline (DPBS) without CaCl2 and MgCl2 (Sigma-Aldrich, catalog number: D8537 )
  17. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  18. ExoQuick-TC (System Biosciences, catalog number: EXOTC10A-1 )
  19. Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
  20. 4,6-Diamidino-2-phenylindole dihydrochloride(DAPI) (Sigma-Aldrich, catalog number: D9542 )
  21. Alexa Fluor-647 conjugated wheat germ agglutinin (WGA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: W32466 )
  22. 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3922 )
  23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  24. Formaldehyde solution (37 wt. % in H2O) (Sigma-Aldrich, catalog number: 252549 )
  25. TritonX-100 (Sigma-Aldrich, catalog number: T8787 )
  26. Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H6648 )
  27. BODIPY stock solution (see Recipes)
  28. Exosome depleted serum (see Recipes)
  29. Fixation solution (see Recipes)
  30. Permeabilization buffer (see Recipes)
  31. WGA conjugate stock solution (see Recipes)

Equipment

  1. Pipette-aid
  2. Water bath
  3. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall ST 8R )
  4. 37 °C with 5% CO2 cell culture incubator
  5. Inverted microscope (Nikon Instruments, model: Eclipse TS100 , Objective: 10x)
  6. SW41Ti rotor (Beckman Coulter, model: SW41Ti )
  7. Zetasizer Nano (Malvern Instruments, model: NanoSight LM10 )
  8. Olympus FV1000 confocal microscope (Olympus, model: FV1000 , Objective:60x)
  9. Biosafety cabinet
  10. Ultracentrifuge (Beckman Coulter, model: L8-80M )

Procedure

  1. Revival of Huh7.5 or Rep2a-Rluc (hepatocytes) cell lines
    1. Rapidly thaw frozen Huh7.5 or Rep2a-Rluc cells (~1 ml) in a 37 °C water bath.
    2. Transfer entire content of Huh7.5 or Rep2a-Rluc cells into a sterile 50 ml conical tube and slowly add 10 ml of pre-warmed DMEM media containing 10% FBS and 100 U/ml of penicillin and streptomycin (complete media).
    3. Mix well, and centrifuge at 300 x g for 10 min at RT.
    4. Discard the supernatant and resuspend cell pellet in 5 ml of complete media and transfer to a T-25 cm2 culture flask.
    5. Incubate cells at 37 °C in a 5% CO2 incubator.
    6. Observe culture flasks routinely under inverted microscope using 10x objective and split cells one day before the experiment.
      Note: Next day after revival, observe the culture flask for floating cells. If floating cells are seen, replace flask with fresh 5 ml of complete media. Hepatoma cells should be split in 1:3 to 1:5 ratio in every 3-5 days to maintain the cell growth.

  2. BODIPY labeling of exosomes
    1. Seed 2 x 106 Huh7.5 cells of ~50% confluency in a 100 mm plate and incubate at 37 °C in 5% CO2 incubator overnight. Next day, infect cells with HCV at a multiplicity of infection (MOI) of 0.1. Allow virus to adsorb onto the cells for 8 h in a minimum volume (2 ml) of DMEM with 2% of FBS and antibiotics. Wash the virus-infected cells 3 times with PBS.
      Note: Media with reduced serum concentration helps in virus infection. PBS should be kept at room temperature before use.
    2. Alternatively, seed 3-4 x 106 Rep2a-Rluc cells of ~80% confluency in a 100 mm plate and incubate cells at 37 °C in 5% CO2 incubator overnight. Next day, wash the cells 3 times with PBS.
    3. Dilute 10 μl of 1 mM of BODIPY 493/503 solution (see Recipes) to 10 ml of culture media to make the final concentration of 1 μM, mix well and add onto the virus infected Huh7.5 or Rep2a-Rluc cells.
    4. Incubate cells for 1 h at 37 °C in 5% CO2 incubator.
    5. Wash the cells 3 times with PBS.
    6. Add 10 ml of fresh DMEM media with 2% of exosome depleted sera (see Recipes) onto the cells and incubate for either 3 days or 24 h for HCV infected Huh7.5 or Rep2a-Rluc cells respectively.
    7. After incubation, collect culture supernatant and proceed for exosomes isolation.

  3. Exosome isolation by ultracentrifugation
    1. Collect 10 ml of culture supernatant from HCV infected Huh7.5 or Rep2a-Rluc cells and centrifuge at 300 x g at 4 °C for 5 min. Without disturbing the cell pellet, carefully transfer the supernatant to a new ultra-clear centrifuge tube.
    2. To get rid of possible cell debris, centrifuge the supernatant at 2,000 x g for 10 min at 4 °C, followed by 26,500 x g for 30 min at 4 °C using SW41 Ti rotor by ultracentrifugation. Transfer the supernatant to a new ultra-clear centrifuge tube.
    3. Then, centrifuge the supernatant using SW41 Ti rotor at 110,000 x g for 90 min at 4 °C by ultracentrifugation to isolate the exosomes. Discard the supernatant.
    4. Wash the exosome pellet 2 times with PBS. For this, resuspend pellet in 10 ml of PBS and centrifuge at 110,000 x g for 60 min at 4 °C. Discard the supernatant. Repeat this step again.
    5. Resuspend the final pellet referred to as exosomes in PBS to 1/20 of the original volume of culture supernatant for analysis.
    6. Check the size distribution of exosomes by dynamic light scattering (DLS) using a Zetasizer Nano (Malvern Instruments). Size distribution analysis of exosomes by DLS is shown in Figure 1. Exosome size is measured as diameter in nm (d. nm) and Polydispersity index (PDI) indicates size distribution of exosomes within the sample.


      Figure 1. Size distribution analysis of purified exosomes by DLS (Nano-ZS90, Malvern). Mean value from the intensity distribution, Z average is 202 nm and polydispersity index, PDI is 0.34.

  4. Exosome isolation by ExoQuick method
    1. Collect 10 ml of culture supernatant from HCV infected Huh7.5 or Rep2a-Rluc cells.
    2. To get rid of possible cell debris, centrifuge at 3,000 x g at 4 °C for 15 min. Transfer the supernatant to a new 15 ml conical tube.
    3. Add ExoQuick at a ratio of 5:1 in the supernatant (5 ml of culture supernatant and 1 ml of ExoQuick), mix well and incubate overnight at 4 °C.
    4. Centrifuge the cell culture supernatant and ExoQuick mixture at 2,000 x g for 30 min at 4 °C.
    5. Remove the residual supernatant and resuspend the final pellet referred to as exosomes in PBS for analysis.

  5. Uptake of BODIPY labeled exosomes
    1. Grow immortalized human hepatic stellate cell lines, LX2 in DMEM media containing 10% FBS and 100 U/ml of antibiotics (complete media).
    2. Pre-coat the 4-well chamber slides with poly-L-lysine (0.01%) for 1 h at 37 °C.
    3. Split LX2 cells and resuspend cell pellet in DMEM media supplemented with 2% exosome depleted FBS and antibiotics.
    4. Seed 1 x 104 LX2 cells in each well of 4-well chamber slides.
    5. Incubate cells at 37 °C in 5% CO2 incubator overnight.
    6. Next day, expose LX2 cells with BODIPY labeled exosomes for 3 h (short) or 24 h (long).
      Note: Exosomes should be diluted in serumfree media from 1:2 to 1:10 before adding onto LX2 cells. Do not add exosomes directly onto the cells. Since exosomes are resuspended in PBS, longer incubation of cells with exosomes will detach cells from chamber slides. To observe any changes at molecular level such as gene expression, exosomes should be incubated with recipient cells for a minimum of 24 h.
    7. After incubation, process LX2 cells for confocal microscopy to observe exosome internalization.

  6. Staining of cells for confocal microscopy
    1. Fix the cells with fixation solution (3.7% formaldehyde, see Recipes) for 20 min at RT.
    2. Wash the cells 3 times with PBS.
    3. Permeabilize cells with permeabilization buffer (0.2% TritonX-100, see Recipes) for 5 min at RT.
    4. Wash the cells 3 times with PBS.
    5. Stain the cell nuclei with DAPI (1 µg/ml) for 2 min at RT (blue color) and observed under a confocal microscope. A representative image is shown in Figure 2A.
    6. For cell boundary or plasma membrane staining, incubate live LX2 cells with 5 µg/ml of Alexa Fluor-647 conjugated WGA (see Recipes) for 10 min at 37 °C.
    7. Wash the cells 2 times with HBSS.
    8. Fix the cells and follow the procedure as mentioned above. A representative image is shown in Figure 2B.


      Figure 2. Internalization of BODIPY labeled exosomes by hepatic stellate cells (LX2). BODIPY labeled exosomes are isolated from the culture supernatants of HCV infected hepatocytes. LX2 cells are exposed with BODIPY labeled exosomes for 3 h. A. Immunofluorescence images showing uptake of BODIPY labeled exosomes (green color) and nuclei (blue color) by LX2 cells. B. Immunofluorescence images showing uptake of BODIPY labeled exosomes (green color), WGA (white color) to mark cell boundary and nuclei (blue color) by LX2 cells. The red arrow indicates BODIPY labeled exosomes inside the cell. The images are taken at 60x2 X magnification.
      Notes:
      1. It is very hard to observe evenness of exosome labeling by microscopy. They are too small to be individually resolved. Generally, under microscope, BODIPY labeled exosomes tend to clump together. Exosomes size vary from 50-150 nm in diameter, however, high protein content in exosomes increases the size up to 200 nm in diameter.
      2. Two commonly used method for exosome isolation worked on different principles. Ultracentrifugation involves differential centrifugation. Successive rounds of centrifugation are intended to pellet down sequentially apoptotic bodies, cell debris, shedding vesicles and then, the exosomes are isolated based on size and density. It is a timeconsuming process involves multiple steps and potentially lead to exosomal aggregation. Exoquick method involves polymer based precipitation technique. There is a chance of contamination of exosomes with microvesicles and apoptotic bodies, however, exosomes can be isolated from very small amount of sample by this method.

Data analysis

Each experiment has been repeated at least three times to verify reproducibility.

Recipes

  1. BODIPY stock solution
    1. Prepare 5 mM (1.25 mg/ml) stock solution in DMSO
    2. Store the stock solution at -20 °C wrapped in aluminum foil
    3. Protect it from light and make smaller aliquots to avoid repeated freeze-thaw
    4. Prepare 1 mM working solution in DMSO at the time of experiment
  2. Exosome depleted serum
    Ultracentrifuge FBS at 110,000 x g at 4 °C for 16 h using SW41 Ti rotor (Beckman Coulter), collect the supernatant and then pass through a 0.22 µm filter
    Store the filtered exosome depleted serum at 4 °C for further use
  3. Fixation solution
    Prepare 3.7% formaldehyde by diluting 37% formaldehyde solution in PBS
  4. Permeabilization buffer
    Add 2 µl of Triton X-100 in 1 ml of PBS
    Always prepare fresh buffer at the time of experiment and vortex it properly
  5. WGA conjugate stock solution
    1. Prepare 1 mg/ml stock solution by dissolving 5 mg of lyophilized powder in 5 ml of HBSS
    2. Store the stock solution at -20 °C wrapped in aluminum foil
    3. Protect it from light and make smaller aliquots to avoid repeated freeze-thaw

Acknowledgments

This protocol is adapted from previously published paper (Devhare et al., 2017). Huh7.5, Rep2a-Rluc and LX2 cell lines are kindly provided by Charles M Rice, Rockefeller University, New York, USA, Hengli Tang, Florida State University, Florida, USA and Scott Friedman, Mount Sinai School of Medicine, NY, USA respectively. The author declares that there is no conflict of interest.

References

  1. Devhare, P. B., Sasaki, R., Shrivastava, S., Di Bisceglie, A. M., Ray, R. and Ray, R. B. (2017). Exosome-mediated intercellular communication between hepatitis C virus-infected hepatocytes and hepatic stellate cells. J Virol 91(6).
  2. Li, J., Liu, K., Liu, Y., Xu, Y., Zhang, F., Yang, H., Liu, J., Pan, T., Chen, J., Wu, M., Zhou, X. and Yuan, Z. (2013). Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity. Nat Immunol 14(8): 793-803.
  3. Qu, Z., Wu, J., Wu, J., Luo, D., Jiang, C. and Ding, Y. (2016). Exosomes derived from HCC cells induce sorafenib resistance in hepatocellular carcinoma both in vivo and in vitro. J Exp Clin Cancer Res 35(1): 159.
  4. Ramakrishnaiah, V., Thumann, C., Fofana, I., Habersetzer, F., Pan, Q., de Ruiter, P. E., Willemsen, R., Demmers, J. A., Stalin Raj, V., Jenster, G., Kwekkeboom, J., Tilanus, H. W., Haagmans, B. L., Baumert, T. F. and van der Laan, L. J. (2013). Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 110(32): 13109-13113.
  5. Schorey, J. S., Cheng, Y., Singh, P. P. and Smith, V. L. (2015). Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO Rep 16(1): 24-43.
  6. Shrivastava, S., Devhare, P., Sujijantarat, N., Steele, R., Kwon, Y. C., Ray, R. and Ray, R. B. (2015). Knockdown of autophagy inhibits infectious hepatitis C virus release by the exosomal pathway. J Virol 90(3): 1387-1396.
  7. Sun, L., Wang, X., Zhou, Y., Zhou, R. H., Ho, W. Z. and Li, J. L. (2016). Exosomes contribute to the transmission of anti-HIV activity from TLR3-activated brain microvascular endothelial cells to macrophages. Antiviral Res 134: 167-171.

简介

外来体已经成为细胞间通讯的重要介质。 它们存在于细胞外环境中,因此可以被邻近或远处的细胞轻易获得。 它们在囊泡内携带mRNA,microRNA和蛋白质,并一度被受体细胞内化; 它们可以通过诱导抗病毒状态向疾病进展调节多种信号传导途径,具有多效性。 我们以前已经显示,携带基因组长度复制子的丙型肝炎病毒(HCV)感染的肝细胞或肝癌细胞在培养上清液中分泌外泌体。 这些外来体被肝星状细胞(HSC)摄取,并在HCV感染期间激活它们以诱导纤维化。 在这里,我们描述了外来体分离和肝星状细胞摄取BODIPY标记的外来体的详细的协议。

【背景】外来体是直径为40-150nm的小的膜结合的细胞外囊泡。已在所有体液中鉴定出它们,包括尿,羊水,血清,唾液,母乳,脑脊液,鼻分泌物和组织培养的细胞系的上清液中。外泌体通过转移微小RNA(mRNA),mRNA和蛋白质来介导细胞与细胞之间的通讯。 其可被邻近或远处的细胞摄取,并随后促进受体细胞中的信号传导(Schorey等人,2015)。外泌体富含蛋白质,包括四跨膜蛋白家族成员(CD9,CD81,CD63),热休克蛋白(Hsp60,Hsp70,Hsp90)和多泡体蛋白(annexins,RabGTPases和内体分选复合物,蛋白质)。最近的研究表明,外来体可以影响免疫应答(Li等人,2013; Sun等人,2016),促进幼稚肝细胞瘤细胞中产生感染(Ramakrishnaiah 等人,2013; Shrivastava等人,2015),疾病进展(Devhare等人,2017),以及抗药物抗性癌症治疗(Qu等人,2016年)。最常用的分离外泌体的技术包括超速离心(UC)和ExoQuick(EQ)沉淀。在丙型肝炎病毒(HCV)感染中,包括肝细胞,巨噬细胞,内皮细胞,淋巴细胞和星状细胞的肝脏驻留细胞之间的串扰在疾病进展中起重要作用。我们之前已经显示,携带基因组长度复制子的HCV感染的肝细胞或肝细胞瘤细胞在细胞培养上清液中分泌外泌体(Shrivastava等人,2015)。这些外泌体携带在肝星状细胞中诱导纤维形成的miR-19a,其可能导致HCV感染期间的肝纤维化(Devhare等人,2017)。在这里,我们提供了一个绿色荧光亲脂性染料,BODIPY标记的exosomes协议实时监测和定位外体在接受肝星状细胞。

关键字:外泌体, 肝星状细胞, BODIPY, 动态光散射, 肝细胞, 丙型肝炎病毒

材料和试剂

  1. 血清移液管(5毫升,10毫升,25毫升)(康宁)
  2. 50ml聚丙烯离心管(Corning,目录号:430829)
  3. T-25厘米2细胞培养瓶(Corning,目录号:430639)
  4. 100mm组织培养物处理的培养皿(Corning,目录号:430167)
  5. 用于SW41Ti转子(Beckman Coulter,目录号:344059)的超纯净离心管,13.2毫升,14×89毫米

  6. 15 ml聚丙烯离心管(Corning,目录号:430791)
  7. 4孔带载玻片(Thermo Fisher Scientific,Nunc,目录号:177437)
  8. 铝箔(Fisher Scientific,Fisherbrand TM,目录号:01-213-102)
  9. 0.2微米大小的过滤器(Acrodisc注射式过滤器)(颇尔,目录号:4612)
  10. Huh7.5细胞系(人肝癌细胞系)
  11. Rep2a-Rluc细胞系:携带丙型肝炎病毒基因组长度复制子的Huh7.5细胞
  12. LX2细胞(永生化人肝星状细胞)
  13. DMEM培养基(Sigma-Aldrich,目录号:D5796)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:16000044)
  15. 青霉素/链霉素(Sigma-Aldrich,目录号:P0781)
  16. 不含CaCl 2和MgCl 2(Sigma-Aldrich,目录号:D8537)的磷酸盐缓冲盐水(DPBS)
  17. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  18. ExoQuick-TC(System Biosciences,目录号:EXOTC10A-1)
  19. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)
  20. 4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D9542)
  21. Alexa Fluor-647共轭小麦胚凝集素(WGA)(Thermo Fisher Scientific,Invitrogen TM,目录号:W32466)
  22. 4,4-二氟-1,3,5,7,8-五甲基-4-硼杂-3a,4α-二氮杂-s-引达省(BODIPY 493/503)(Thermo Fisher Scientific,Invitrogen TM TM >,目录号:D3922)
  23. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  24. 甲醛溶液(在H 2 O中37重量%)(Sigma-Aldrich,目录号:252549)
  25. TritonX-100(Sigma-Aldrich,目录号:T8787)
  26. Hanks平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H6648)
  27. BODIPY原液(见食谱)
  28. Exosome耗尽血清(见食谱)
  29. 固定解决方案(见食谱)
  30. 渗透缓冲液(见食谱)
  31. WGA结合物原液(见食谱)

设备

  1. 移液助剂
  2. 水浴
  3. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall ST 8R)
  4. 37℃,5%CO 2细胞培养箱培养
  5. 倒置显微镜(尼康仪器,型号:Eclipse TS100,目标:10x)
  6. SW41Ti转子(Beckman Coulter,型号:SW41Ti)
  7. Zetasizer Nano(Malvern Instruments,型号:NanoSight LM10)
  8. 奥林巴斯FV1000共聚焦显微镜(奥林巴斯,型号:FV1000,目标:60x)
  9. 生物安全柜
  10. 超速离心机(Beckman Coulter,型号:L8-80M)

程序

  1. Huh7.5或Rep2a-Rluc(肝细胞)细胞系的复活
    1. 在37℃水浴中快速融化冷冻的Huh7.5或Rep2a-Rluc细胞(〜1ml)。
    2. 将Huh7.5或Rep2a-Rluc细胞的全部内容物转移到无菌的50ml锥形管中,并缓慢加入10ml预热的含有10%FBS和100U / ml青霉素和链霉素(完全培养基)的DMEM培养基。 />
    3. 充分混合,并在室温下300×g离心10分钟。
    4. 弃去上清,用5ml完全培养基重悬细胞沉淀,转移到T-25cm 2培养瓶中。

    5. 培养细胞在37°C在5%CO 2孵化器。

    6. 在倒置显微镜下使用10倍物镜观察培养瓶,并在实验前一天分开细胞。
      注意:复苏后的第二天,观察培养瓶中的浮游细胞。如果看到漂浮的细胞,用新鲜的5毫升完全培养基替换烧瓶。肝细胞瘤细胞应每3-5天以1:3至1:5的比例分裂以维持细胞生长。

  2. BODIPY标签的外来体
    1. 在100mm平板中播种约50%融合的2×10 6 Huh7.5细胞并在37℃下在5%CO 2培养箱中培养过夜。第二天,感染复数(MOI)为0.1的HCV感染细胞。在含有2%FBS和抗生素的最小体积(2ml)的DMEM中,让病毒吸附到细胞上8小时。用PBS清洗病毒感染的细胞3次。
      注意:血清浓度降低的培养基有助于病毒感染。
      在使用PBS前,应将PBS保存在室温下
    2. 或者,在100mm平板中接种约80%融合的种子3-4×10 6 Rep2a-Rluc细胞,并在37℃下在5%CO 2培养箱过夜。第二天,用PBS清洗细胞3次。
    3. 将10μl1mM BODIPY 493/503溶液(见配方)稀释到10ml培养基中,使最终浓度为1μM,充分混合并加到病毒感染的Huh7.5或Rep2a-Rluc细胞上。 />

    4. 在37℃,5%CO 2培养箱中孵育细胞1小时
    5. 用PBS清洗细胞3次。
    6. 添加10毫升的新鲜DMEM介质与2%的exosome耗尽血清(见食谱)到细胞上,分别为HCV感染的Huh7.5或Rep2a-Rluc细胞孵育3天或24小时。
    7. 孵育后,收集培养上清液并进行exosomes分离。

  3. 通过超速离心分离外来体
    1. 收集来自HCV感染的Huh7.5或Rep2a-Rluc细胞的10ml培养物上清液,并在300℃下在4℃下离心5分钟。在不干扰细胞沉淀的情况下,小心地将上清液转移到新的超纯净离心管中。
    2. 为了除去可能的细胞碎片,将上清液在4℃下以2,000xg离心10分钟,然后在4℃下用26,500xg离心30分钟,使用SW41 Ti转子超速离心。将上清液转移到一个新的超纯净离心管中。
    3. 然后,使用SW41T1转子,在4℃下,通过超速离心将上清液在110,000×gg下离心90分钟以分离外泌体。丢弃上清。
    4. 用PBS洗涤外来体小球2次。为此,将沉淀重悬于10ml PBS中并在4℃下以110,000×gg离心60分钟。丢弃上清液。重复这一步。
    5. 将称为外来体的PBS中的最终沉淀重悬于原始体积的培养上清液的1/20用于分析。
    6. 使用Zetasizer Nano(Malvern Instruments)通过动态光散射(DLS)检查外泌体的大小分布。通过DLS的外泌体的尺寸分布分析显示在图1中。外体尺寸以nm(d.nm)的直径测量,多分散性指数(PDI)指示样品内exosome的尺寸分布。

      图1.通过DLS(Nano-ZS90,Malvern)纯化的外来体的尺寸分布分析。从强度分布的平均值来看,Z平均值为202nm,多分散指数PDI为0.34。

  4. 用ExoQuick方法分离外来体
    1. 从HCV感染的Huh7.5或Rep2a-Rluc细胞收集10毫升的培养上清液。
    2. 为了除去可能的细胞碎片,在4℃以3000×g离心15分钟。将上清液转移到新的15ml锥形管中。
    3. 在上清液(5毫升培养上清液和1毫升ExoQuick)中以5:1的比例添加ExoQuick,充分混合并在4°C孵育过夜。
    4. 在4℃将细胞培养上清液和ExoQuick混合物在2000gxg离心30分钟。
    5. 去除残留的上清液,并重悬在PBS中称为外来体的最终颗粒进行分析。

  5. BODIPY标记的外泌体的摄取
    1. 在含有10%FBS和100U / ml抗生素(完全培养基)的DMEM培养基中培养永生化人肝星状细胞系LX2。

    2. 用聚-L-赖氨酸(0.01%)在37℃预涂覆4孔室玻片1小时。
    3. 拆分LX2细胞,并在补充有2%exosome耗尽的FBS和抗生素的DMEM培养基中重悬细胞沉淀。
    4. 在4孔室玻片的每个孔中种入1×10 4个LX2细胞。

    5. 在37℃,5%CO 2培养箱中孵育细胞过夜
    6. 第二天,用BODIPY标记的外泌体暴露LX2细胞3小时(短)或24小时(长)。
      注意:在加入到LX2细胞中之前,外泌体应在无血清培养基中从1:2稀释到1:10。不要将外来体直接添加到细胞上。由于外体被重新悬浮于PBS中,细胞与外体的更长时间的孵育将使细胞从腔室载玻片上分离。为了观察分子水平上的任何变化,如基因表达,应将外泌体与受体细胞一起孵育至少24小时。
    7. 温育后,处理LX2细胞共聚焦显微镜观察外来体内化。

  6. 细胞染色共聚焦显微镜

    1. 用固定液(3.7%甲醛,见配方)固定细胞20分钟
    2. 用PBS清洗细胞3次。
    3. 透化缓冲液(0.2%TritonX-100,见配方)在室温下透化细胞5分钟。
    4. 用PBS清洗细胞3次。
    5. 在室温(蓝色)下用DAPI(1μg/ ml)染色细胞核2分钟,并在共聚焦显微镜下观察。图2A中显示了代表性的图像。
    6. 对于细胞边界或质膜染色,用5μg/ ml Alexa Fluor-647缀合的WGA(参见配方)在37℃孵育活的LX2细胞10分钟。
    7. 用HBSS清洗细胞2次。
    8. 修复细胞,并按照上述过程。代表性的图像如图2B所示。


      图2.肝星状细胞(LX2)对BODIPY标记的外泌体的内化。 BODIPY标记的外泌体从HCV感染的肝细胞的培养上清液中分离。用BODIPY标记的外泌体暴露LX2细胞3小时。 A.显示LX2细胞摄取BODIPY标记的外来体(绿色)和细胞核(蓝色)的免疫荧光图像。 B.显示BODIPY标记的外来体(绿色),WGA(白色)被LX2细胞标记细胞边界和细胞核(蓝色)的摄取的免疫荧光图像。红色箭头表示BODIPY标记的细胞内的外泌体。这些图像是以60x2的放大倍数拍摄的。
      注意:
      1. 通过显微镜观察外来体标记的均匀性是非常困难的。他们太小,不能单独解决。通常,在显微镜下,BODIPY标记的外泌体倾向于聚集在一起。外泌体大小直径从50-150nm不等,然而,外泌体中的高蛋白质含量将直径增加到200nm。
      2. 两种常用的外来体分离方法的工作原理不同。超速离心涉及差速离心。连续循环的离心是为了连续沉淀凋亡小体,细胞碎片,脱落小泡,然后根据大小和密度分离外泌体。这是一个耗费时间的过程,涉及多个步骤并可能导致外泌体聚集。 Exoquick方法涉及基于聚合物的沉淀技术。有可能是外来体被微泡和凋亡小体污染,然而,通过这种方法可以从非常少量的样品中分离出外来体。

数据分析

每个实验重复至少三次以验证重现性。

食谱

  1. BODIPY原液
    1. 准备在DMSO中的5毫米(1.25毫克/毫升)原液。
    2. 将原液储存在-20℃的铝箔包装中
    3. 保护它免受光照,并制作更小的等分试样以避免反复冻融
    4. 在实验时准备在DMSO中的1 mM工作溶液
  2. Exosome耗尽血清
    使用SW41Ti转子(Beckman Coulter)在4℃下以110,000×gg离心FBS 16小时,收集上清液,然后通过0.22μm过滤器。
    将过滤的exosome耗尽的血清储存在4°C以备将来使用
  3. 固定解决方案

    用PBS稀释37%的甲醛溶液制备3.7%的甲醛
  4. 透化缓冲液
    加入2μl的Triton X-100在1 ml的PBS中 在实验的时候一定要准备新鲜的缓冲液,并将其正确旋转。
  5. WGA结合物储备溶液
    1. 通过将5mg冻干粉溶解在5ml HBSS中制备1mg / ml储备液。
    2. 将原液储存在-20℃的铝箔包装中
    3. 保护它免受光照,并制作更小的等分试样以避免反复冻融

致谢

该协议是从以前发表的论文(Devhare等人,2017年)改编的。 Huh7.5,Rep2a-Rluc和LX2细胞系由美国纽约洛克菲勒大学的Charles M Rice,美国佛罗里达州佛罗里达州立大学的Hengli Tang和美国纽约西奈山医学院的Scott Friedman提供分别。作者声明不存在利益冲突。

参考

  1. Devhare,P.B.,Sasaki,R.,Shrivastava,S.,Di Bisceglie,A.M.,Ray,R.和Ray,R.B.(2017)。 丙型肝炎病毒感染的肝细胞与肝星状细胞之间的外来体介导的细胞间通讯 J Vir 91(6)。
  2. Li,J.,Liu,K.,Liu,Y.,Xu,Y.,Zhang,F.,Yang,H.,Liu,J.,Pan,T.,Chen,J.,Wu, Zhou X.和Yuan Z.(2013)。 外泌体介导IFN-α诱导的抗病毒活性的细胞间传递。 Nat Immunol 14(8):793-803。
  3. Qu,Z.,Wu,J.,Wu,J.,Luo,D.,Jiang,C。和Ding,Y。(2016)。 来源于HCC细胞的外泌体在肝细胞癌体内均可诱导索拉非尼耐药性 和体外。 J Exp Clin Cancer Res 35(1):159.
  4. 罗摩克里希亚,V.,Thumann,C.,Fofana,I.,Habersetzer,F.,Pan,Q.,de Ruiter,PE,Willemsen,R.,Demmers,JA,Stalin Raj,V.,Jenster, Kwekkeboom,J.,Tilanus,HW,Haagmans,BL,Baumert,TF和van der Laan,LJ(2013)。 外泌体介导的丙型肝炎病毒传染人肝癌Huh7.5细胞。美国国立科学院美国科学院 110(32):13109-13113。
  5. Schorey,J.S.,Cheng,Y.,Singh,P.P.and Smith,V.L。(2015)。 在宿主 - 病原体相互作用中的外泌体和其他细胞外囊泡 EMBO Rep 16(1):24-43。
  6. Shrivastava,S.,Devhare,P.,Sujijantarat,N.,Steele,R.,Kwon,Y.C.Ray,R.and Ray,R.B。(2015)。 敲除自噬可以通过外泌体途径抑制传染性丙型肝炎病毒的释放。 J Virol 90(3):1387-1396。
  7. Sun,L.,Wang,X.,Zhou,Y.,Zhou,R.H.,Ho,W.Z.和Li,J.L。(2016)。 外泌体有助于从TLR3激活的脑微血管内皮细胞向巨噬细胞传递抗HIV活性。 抗病毒药物 134:167-171。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shrivastava, S. (2017). Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells. Bio-protocol 7(23): e2633. DOI: 10.21769/BioProtoc.2633.
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