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Nov 2018

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Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine
使用成像流式细胞术表征从细胞培养基,血浆或尿液分离出的细胞外囊泡   

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Abstract

The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry method for use with an imaging flow cytometer (Amnis FlowSight) for identifying EV released by specific cell types and excreted into the urine or blood using markers characteristic of particular cells in the kidney. Here we present a protocol utilizing the Amnis FlowSight Imaging Flow Cytometer to identify and quantify EV from the urine of patients with essential hypertension and renovascular disease. Notably, EV isolated from cell culture media and plasma can also be analyzed similarly.

Keywords: Exosomes (外来体), Extracellular vesicles (细胞外囊泡), Imaging flow cytometry (成像流式细胞仪), Biomarkers (生物标计), Hypertension (高血压), Renovascular disease (肾血管疾病)

Background

Extracellular vehicles (EVs) are released from cells under normal conditions and their numbers are known to increase when the cells are exposed to stress conditions. Therefore, levels of urinary EVs have been shown to be associated with various kidney disorders such as polycystic kidney disease (Hogan et al., 2009), acute kidney injury (Aghajani Nargesi et al., 2017; Cappuccilli et al., 2018), and various glomerular diseases (Zhang et al., 2019).

We have previously shown that levels of EVs that are increased in patients with hypertension likely originate from podocytes (Kwon et al., 2017) and peritubular capillaries (PTC) (Sun et al., 2018; Zhang et al., 2019). We have also recently shown that urinary levels of EVs reflecting renal cellular senescence are altered in these patients (Santelli et al., 2019). We describe here a more detailed protocol that can be used for the isolation and quantitative characterization of EVs isolated from cell culture media, plasma, and urine (Kwon et al., 2017; Sun et al., 2018; Conley et al., 2019; Zhang et al., 2019). The primary advantages of this method of EV isolation are shorter processing time and obviating the requirement for an ultracentrifuge to isolate the EVs.

Materials and Reagents

  1. Opaque Black 1.5 ml microcentrifuge tubes (Midwest Scientific, catalog number: T7100BK)
  2. Avant clear, 1.5 ml microcentrifuge tubes (Midwest Scientific, catalog number: AVSS1700)
  3. dPBS (Life Technologies, catalog number: 14190-250)
  4. Albumin, bovine serum, fraction V (Sigma, catalog number: A3059)
  5. InvitrogenTM Total Exosome Isolation reagents
    1. From Urine (Thermo Fisher Scientific, Inc., catalog number: 4484452)
    2. From Cell culture media (Thermo Fisher Scientific, Inc., catalog number: 4478359)
    3. From Plasma (Thermo Fisher Scientific, Inc., catalog number: 4484450)
    4. From Serum (Thermo Fisher Scientific, Inc., catalog number: 4478360)
  6. Tag-It VioletTM proliferation and cell tracking dye (Biolegend, Inc., catalog number: 425101) 
  7. PV1/PL-VAP, FITC, anti-human antibody (Lifespan Biosciences, catalog number: LS-C209607-100)
  8. CD31, Brilliant Violet 605, Anti-Human antibody (BioLegend, Inc., catalog number: 303122)
  9. CD144, PE, anti-human antibody (BioLegend, Inc., catalog number: 348506)
  10. Bleach, Clorox (Solution used in the FlowSight during operation)
  11. Isopropanol, HPLC grade (Honeywell, catalog number: AH323-4) (Solution used in the FlowSight during operation)
  12. Coulter Clenz® (BD Biosciences, catalog number: 8546929) (Solution used in the FlowSight during operation)
  13. Deionized H2O (Solution used in the FlowSight during operation)
  14. Molecular ProbesTM AbC Total Antibody compensation bead kit (Thermo Fisher Scientific, Inc., catalog number: A10497)
  15. Sucrose (Sigma, BioXtra, catalog number: S7903)
  16. MES: 2-(N-Morpholino) ethanesulfonic acid, 4-Morpholineethanesulfonic acid (Sigma, catalog number: M3671)
  17. DMSO
  18. EV Buffer (pH 7.4) (see Recipes)
  19. 5 mM Tag-It VioletTM (TIV) solution (see Recipes)

Equipment

  1. 1 ml pipettor
  2. FlowSight Imaging flow cytometer (Amnis, Inc.) equipped with:
    1. 488 nm Laser
    2. 405 nm Laser
    3. 642 nm laser
    4. 785 nm laser (SSC)
    5. Quantitative imaging
    6. 96 well plate autosampler
  3. Refrigerated Microcentrifuge (Eppendorf, Model 5415R, 48 x 2 ml rotor)
  4. MultiTherm incubated vortexer (Midwest Scientific, catalog number: MTHC-1500)
  5. Barnstead deionized water system (Model)
  6. -80 °C freezer
  7. -20 °C freezer
  8. 4 °C refrigerator

Software

  1. Amnis® IDEAS version 6.2 (Luminex Inc., https://www.luminexcorp.com/imaging-flow-cytometry/), data analysis software
  2. Amnis® INSPIRE Version 6, data acquisition software
  3. Excel (Microsoft)
  4. JMP version 10.0 (SAS Institute)

Procedure

  1. EV isolation
    Note: EVs were isolated from whole urine using Total Exosome Isolation reagent (Invitrogen, Waltham, MA) according to the manufacturer’s guidelines. 
    1. Urine samples (1,000 μl) were centrifuged at 2,000 x g for 30 min at 4 °C to remove cells and debris.
    2. Urine supernatants (800 μl) were mixed with 1 volume (800 μl) of the Total Exosome Isolation reagent and incubated for 1 h at room temperature.
      Other samples (Plasma, serum, cell culture media) can be prepared similarly using the manufacturer’s recommendations for reagent volume, incubation time, and temperature.
    3. After incubation, samples were centrifuged at 10,000 x g for 1 h at 4 °C. 
    4. Supernatant was removed using 1 ml pipettor and discarded.
    5. Pelleted exosomes were resuspended in 50 μl EV buffer (2 mol/L sucrose and 500 mmol/L MES [2-(N-morpholino) ethanesulfonic acid, 4-morpholineethanesulfonic acid] solution).
    6. Store at -80 °C. Samples are considered to be stable for at least 6 months (Mendt et al., 2018).

  2. EV staining and antibody labeling
    1. Start up and Calibrate FlowSight.
    2. Thaw samples on ice.
    3. Prepare 5 mM solution of Tag-It VioletTM by adding 50 μl DMSO to one tube of Tag-It VioletTM as in the Recipe 2 Tag-It Violet marks the EV by binding to the interior proteins, thus labeling the EVs, which are too small for brightfield visualization. 
    4. Pipet 20 μl EVs using 20 μl pipettor into 1.5 ml opaque Black microcentrifuge tubes, to protect samples from light. The incubated vortexer has a clear lid and is often located in the main laboratory, thus the black tubes protect the fluorescent dyes from photobleaching during the incubations.
      Include one sample of EVs that will be stained only with Tag-It Violet for single color compensation control.
    5. Add 30 μl dPBS to each sample using a 100 μl pipettor.
    6. Add 0.5 μl of 5 mM TIV stock to EVs (50 μM final concentration) using a 2 μl pipettor.
    7. Incubate EV for 2 h at 37 °C at 250 rpm using MultiTherm incubated vortexer. 
    8. Add antibodies (for example peritubular capillary identification) using a 10 μl pipettor.
      3 μl PL-VAP(LSBio)
      4 μl CD31 (Biolegend)
      3 μl CD144 (Biolegend)
      Incubate for 1 h at RT at 250 rpm in MultiTherm incubated vortexer.

  3. Data acquisition using FlowSight
    1. Acquisition is performed using FlowSight Imaging Flow Cytometer (Amnis Corporation, Seattle, WA) equipped with Amnis INSPIRE` software. 
    2. Vortex sample for 10-20 s before loading onto FlowSight.
    3. Instrument setup:
      Lasers (all set to full power):
      405 nm 100 mW
      488 nm 60 mW
      642 nm 100 mW
      785 nm (SSC) 70 mW
    4. Acquire at least 100,000 TIV positive events.
    5. Prepare single color control compensation beads for antibodies.
      1. In 1.5 ml microcentrifuge tubes add 1 drop AbC capture beads.
      2. Add one antibody per tube with volume indicated in Step B8.
      3. Incubate at Room Temp for 15 min protected from light.
      4. After adding 1 ml dPBS, vortex and spin at 250 x g for 5 min using a 1000 μl pipettor.
      5. Remove supernatant.
      6. Add one drop AbC negative beads and 50 μl dPBS.
      7. Vortex and run on FlowSight.
        1. Acquire 1000 events with Brightfield off (for compensation).
        2. Acquire 1000-5000 events with Brightfield on to aid in initial gate determination.
    6. Samples are run in the following order.
      1. TIV only EVs
        1. Acquire 1000 events with Brightfield off (for compensation).
        2. Acquire 1000-5000 events with Brightfield on to aid in initial gate determination.
      2. Antibody single color controls (acquired as in Step C5g above)
      3. Samples with TIV and all antibodies (Brightfield is on)

Data analysis

  1. Amnis IDEAS data analysis software
    1. EVs were quantified using IDEAS (Amnis, Seattle, WA). 
    2. Compensation matrix for experiment is created using IDEAS compensation matrix wizard.
    3. Raw data file from one sample is opened and compensation matrix applied.
    4. Gating strategy template is created using stepwise data analysis wizard in IDEAS software based on antibody and Tag-It Violet staining panel. Figure 1 illustrates the gating strategy steps taken in flow cytometry data analysis for PTC identification.
      For PTC identification, Tag-It Violet+/PL-VAP+/CD31-/CD144- events were of primary interest and the gating strategy was developed with that goal. The combination of PL-VAP+/CD31-/CD144- is used for identification of EV released from peritubular capillaries.


      Figure 1. A schematic showing flow cytometry gating strategy for PTC identification. Step 1: Scatterplot of side scatter (size) and intensity of Tag-It Violet (EVs) from all events collected, Draw gate (ROI) for Tag-It violet positive events and SSC low; Step 2: Histogram of PL-VAP antibody intensity of the EV+ population, draw gate (ROI) for PL-VAP positive events; Step 3: Scatterplot of CD31 and CD144 from PL-VAP+ population, most important gate (ROI) being CD31-/CD144- for PTC EV, however gates (ROIs) for identification of CD31-/CD144+, CD31+, CD144-, and CD31+/CD144+ events are also drawn.

    5. EVs are measured using the following flow-gating strategy (see Figure 1).
      1. Create Scatterplot of Side-scatter (SSC) channel 6 versus Tag-It Violet channel 7 of all events collected.
        1. Draw a region of interest (ROI) for positive Tag-it violet events and low SSC
        2. Label as Tag-It Violet+ or EV+ population
      2. Create Histogram of PL-VAP channel. 
        1. Draw an ROI for positive PL-VAP
        2. Label as PL-VAP+
      3. Create Scatterplot of CD31 versus CD144
        1. Draw an ROI around events that are for negative CD31 and negative CD144 
        2. Label as CD31-/CD144-
        3. Also draw ROIs for CD31+/CD144+ events
    6. Gating strategy with all ROIs is saved as a template within IDEAS.
      1. Saved template and compensation matrix is applied to all samples.
      2. Manual verification of ROIs on 3-5 samples was done to verify ROI border thresholds
      3. Data as number of events in each ROI from every sample was extracted.
    7. The gated populations are expressed as a percentage of Tag-It Violet positive events. (See Sun et al. (2018), Figures 2A and 2C, for comparison of data between groups of patients.)
      1. PL-VAP+/CD31-/CD144- events
      2. PL-VAP+/CD31+
      3. PL-VAP+/CD144+
      4. PL-VAP+/CD31+/CD144+

  2. Statistical analysis using JMP; please see Sun et al. (2018) for data analysis.

Recipes

  1. EV Buffer (pH 7.4)
    2 mol/L sucrose (Sigma, BioXtra)
    500 mmol/L MES (Sigma)
    Stable for 6 months at 4 °C
  2. 5 mM Tag-It VioletTM (TIV) solution
    50 μl anhydrous DMSO
    One vial of Tag-It VioletTM (122.25 μg)
    Stable for 1 month at -20 °C, but best practice is to make fresh

Acknowledgments

This protocol was adapted from Sun et al. (2018). This work was partly supported by the National Institutes of Health grant numbers DK10081, DK104273, DK102325, and DK120292.

Competing interests

No competing interests.

Ethics

This study was approved by the institutional review board of the Mayo Clinic, and performed in accordance with the ethical principles of the Declaration of Helsinki. Informed written consent was obtained from each patient.

References

  1. Aghajani Nargesi, A., Lerman, L. O. and Eirin, A. (2017). Mesenchymal stem cell-derived extracellular vesicles for kidney repair: current status and looming challenges. Stem Cell Res Ther 8(1): 273. 
  2. Cappuccilli, M., Capelli, I., Comai, G., Cianciolo, G. and La Manna, G. (2018). Neutrophil gelatinase-associated lipocalin as a biomarker of allograft function after renal transplantation: evaluation of the current status and future insights. Artif Organs 42(1): 8-14.
  3. Conley, S. M., Shook, J. E., Zhu, X. Y., Eirin, A., Jordan, K. L., Woollard, J. R., Isik, B., Hickson, L. J., Puranik, A. S., and Lerman, L. O. (2019). Metabolic syndrome induces release of smaller extracellular vesicles from porcine mesenchymal stem cells. Cell Transplant (unpublished).
  4. Hogan, M. C., Manganelli, L., Woollard, J. R., Masyuk, A. I., Masyuk, T. V., Tammachote, R., Huang, B. Q., Leontovich, A. A., Beito, T. G., Madden, B. J., Charlesworth, M. C., Torres, V. E., LaRusso, N. F., Harris, P. C. and Ward, C. J. (2009). Characterization of PKD protein-positive exosome-like vesicles. J Am Soc Nephrol 20(2): 278-288. 
  5. Kwon, S. H., Woollard, J. R., Saad, A., Garovic, V. D., Zand, L., Jordan, K. L., Textor, S. C. and Lerman, L. O. (2017). Elevated urinary podocyte-derived extracellular microvesicles in renovascular hypertensive patients. Nephrol Dial Transplant 32(5): 800-807.
  6. Mendt, M., Kamerkar, S., Sugimoto, H., McAndrews, K. M., Wu, C. C., Gagea, M., Yang, S., Blanko, E. V. R., Peng, Q., Ma, X., Marszalek, J. R., Maitra, A., Yee, C., Rezvani, K., Shpall, E., LeBleu, V. S., and Kalluri, R. (2018) Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 3(8):e99263.
  7. Santelli, A., Sun, I. O., Eirin, A., Abumoawad, A. M., Woollard, J. R., Lerman, A., Textor, Stephen C., Puranik, A. S. and Lerman, L. O. (2019). Senescent kidney cells in hypertensive patients release urinary extracellular vesicles. J Am Heart Assoc 8(11): e012584.
  8. Sun, I. O., Santelli, A., Abumoawad, A., Eirin, A., Ferguson, C. M., Woollard, J. R., Lerman, A., Textor, S. C., Puranik, A. S. and Lerman, L. O. (2018). Loss of renal peritubular capillaries in hypertensive patients is detectable by urinary endothelial microparticle levels. Hypertension 72(5): 1180-1188.
  9. Zhang, L. H., Zhu, X. Y., Eirin, A., Nargesi, A. A., Woollard, J. R., Santelli, A., Sun, I. O., Textor, S. C. and Lerman, L. O. (2019). Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria. Am J Physiol Renal Physiol 317(7): F12-F22.

简介

非侵入性地检测对肾脏的特定损伤的能力受到限制。鉴定由细胞释放的细胞外囊泡,尤其是在胁迫下,可能有助于监测和鉴定肾脏内受压的特定细胞类型。我们已经调整了以前发布的传统流式细胞仪方法,与成像流式细胞仪(Amnis FlowSight)配合使用,以使用特定细胞类型的特征性肾脏标志物鉴定由特定细胞类型释放并排泄到尿液或血液中的EV。在这里,我们介绍了一种利用Amnis FlowSight成像流式细胞仪来从原发性高血压和肾血管疾病患者的尿液中鉴定和量化EV的方案。值得注意的是,从细胞培养基和血浆中分离出的EV也可以进行类似的分析。
【背景】
在正常条件下,细胞外载体(EVs)会从细胞中释放出来,并且当细胞暴露于应激条件下时,其数量会增加。因此,尿液电动汽车的水平已被证明与多种肾脏疾病有关,例如多囊肾(Hogan et al。,2009),急性肾损伤(Aghajani Nargesi et al。,2017年; Cappuccilli 等人,,2018年),以及各种肾小球疾病(Zhang 等人,,2019年)。 先前我们已经证明,高血压患者的EV升高水平可能源自足细胞(Kwon等,2017)和肾小管周围毛细血管(PTC)(等)。 em>,2018; Zhang et al。,2019)。我们最近还显示,这些患者的反映肾脏细胞衰老的电动汽车尿液水平发生了变化(Santelli et al。,2019)。我们在这里描述了一种更详细的协议,可用于分离和定量表征从细胞培养基,血浆和尿液中分离出的电动汽车(Kwon等,2017; Sun等) ,2018年;康利等人,,2019年;张等人,,2019年)。这种电动汽车隔离方法的主要优点是处理时间更短,并且无需使用超速离心机来隔离电动汽车。

关键字:外来体, 细胞外囊泡, 成像流式细胞仪, 生物标计, 高血压, 肾血管疾病

材料和试剂

  1. 黑色不透明1.5 ml微量离心管(Midwest Scientific,目录号:T7100BK)
  2. Avant透明1.5毫升微量离心管(Midwest Scientific,目录号:AVSS1700)
  3. dPBS(Life Technologies,目录号:14190-250)
  4. 白蛋白,牛血清,馏分V(西格玛,目录号:A3059)
  5. Invitrogen TM 总外泌体分离试剂
    1. 从尿液中(Thermo Fisher Scientific,Inc.,目录号:4484452)
    2. 来自细胞培养基(Thermo Fisher Scientific,Inc.,目录号:4478359)
    3. 从等离子(Thermo Fisher Scientific,Inc.,目录号:4484450)
    4. 从血清(Thermo Fisher Scientific,Inc.,目录号:4478360)
  6. Tag-It Violet TM 增殖和细胞跟踪染料(Biolegend,Inc.,目录号:425101)
  7. PV1 / PL-VAP,FITC,抗人抗体(Lifespan Biosciences,目录号:LS-C209607-100)
  8. CD31,亮紫605,抗人类抗体(BioLegend,Inc.,目录号:303122)
  9. CD144,PE,抗人抗体(BioLegend,Inc.,目录号:348506)
  10. 漂白剂,Clorox(运行期间在FlowSight中使用的解决方案)
  11. 异丙醇,HPLC级(Honeywell,目录号:AH323-4)(操作期间在FlowSight中使用的溶液)
  12. Coulter Clenz ®(BD Biosciences,目录号:8546992)(操作期间在FlowSight中使用的解决方案)
  13. 去离子H 2 O(操作期间在FlowSight中使用的溶液)
  14. Molecular Probes TM AbC总抗体补偿微珠试剂盒(Thermo Fisher Scientific,Inc.,目录号:A10497)
  15. 蔗糖(Sigma,BioXtra,目录号:S7903)
  16. MES:2-(N-Morpholino)乙磺酸,4-Morpholineethanesulfonic酸(Sigma,目录号:M3671)
  17. 二甲基亚砜
  18. EV缓冲液(pH 7.4)(请参见配方)
  19. 5 mM Tag-It Violet TM (TIV)解决方案(请参阅食谱)

设备

  1. 1毫升移液器
  2. FlowSight Imaging流式细胞仪(Amnis,Inc.)配备:
    1. 488 nm激光
    2. 405 nm激光
    3. 642 nm激光
    4. 785 nm激光(SSC)
    5. 定量成像
    6. 96孔板自动进样器
  3. 冷冻微量离心机(Eppendorf,5415R型,48 x 2 ml转子)
  4. MultiTherm温育涡旋仪(Midwest Scientific,目录号:MTHC-1500)
  5. 巴恩斯特德去离子水系统(型号)
  6. -80°C冷冻室
  7. -20°C冷冻室
  8. 4°C冰箱

软件

  1. Amnis ® IDEAS版本6.2(Luminex Inc., https:// www .luminexcorp.com / imaging-flow-cytometry / ),数据分析软件
  2. Amnis ® INSPIRE版本6,数据采集软件
  3. Excel(微软)
  4. JMP版本10.0(SAS研究所)

程序

  1. 电动汽车隔离
    注意:根据制造商的指导,使用总外泌体分离试剂(Invitrogen,Waltham,MA)从全尿中分离出EV。
    1. 将尿液样品(1,000μl)在4°C下于2,000 x g 离心30分钟,以去除细胞和碎片。
    2. 将尿液上清液(800μl)与1体积(800μl)的总外泌体分离试剂混合,并在室温下孵育1小时。
      其他样品(血浆,血清,细胞培养基)也可以使用制造商关于试剂量,孵育时间和温度的建议类似地制备。
    3. 孵育后,将样品在4°C下以10,000 x g 离心1小时。
    4. 用1 ml移液器移出上清液并丢弃。
    5. 将沉淀的外泌体重悬于50μlEV缓冲液(2 mol / L蔗糖和500 mmol / L MES [2-(N-吗啉代)乙磺酸,4-吗啉乙烷磺酸]溶液)中。
    6. 储存在-80°C。样品被认为至少可以稳定6个月(Mendt et al。,2018)。

  2. EV染色和抗体标记
    1. 启动并校准FlowSight。
    2. 在冰上解冻样品。
    3. 如配方2 Tag-It Violet标记EV一样,通过向一管Tag-It Violet TM 中添加50μlDMSO,制备5 mM Tag-It Violet TM 溶液。通过与内部蛋白质结合,从而标记电动汽车,这些电动汽车对于明场可视化来说太小了。
    4. 使用20μl移液器将20μlEV移液至1.5 ml不透明的黑色微量离心管中,以保护样品免受光照。孵育后的涡旋仪有一个清晰的盖子,通常位于主实验室中,因此黑色试管可以防止荧光染料在孵育过程中发生光漂白。
      包括一个仅用Tag-It Violet染色的EV样本,以进行单色补偿控制。
    5. 使用100微升移液器向每个样品中添加30微升dPBS。
    6. 使用2μl移液器将0.5μl的5 mM TIV储备液添加至EV(最终浓度为50μM)。
    7. 使用MultiTherm温育涡旋仪在37°C和250 rpm下温育EV 2小时。
    8. 使用10μl移液器添加抗体(例如,肾小管周围毛细血管识别)。
      3μlPL-VAP(LSBio)
      4μlCD31(生物传奇)
      3微升CD144(Biolegend)
      在MultiTherm温育涡旋仪中以250 rpm在室温温育1小时。

  3. 使用FlowSight进行数据采集
    1. 使用配备有Amnis INSPIRE`软件的FlowSight成像流式细胞仪(华盛顿州西雅图的安尼斯公司)进行采集。
    2. 在加载到FlowSight之前将样品涡旋10-20 s。
    3. 仪器设置:
      激光器(全部设置为全功率):
      405 nm 100毫瓦
      488 nm 60兆瓦
      642 nm 100毫瓦
      785纳米(SSC)70毫瓦
    4. 至少获得100,000个TIV阳性事件。
    5. 准备用于抗体的单色控制补偿珠。
      1. 在1.5 ml微量离心管中,加入1滴AbC捕获珠。
      2. 每管加入一种抗体,步骤B8中指示的体积。
      3. 在室温下孵育15分钟,避光。
      4. 加入1 ml dPBS后,涡旋并使用1000μl移液器以250 x g 旋转5分钟。
      5. 除去上清液。
      6. 加入一滴AbC阴性珠和50μldPBS。
      7. 涡流并在FlowSight上运行。
        1. 关闭Brightfield以获得1000个事件(用于补偿)。
        2. 开启Brightfield可以获取1000-5000个事件,以帮助初步确定门。
    6. 样本按以下顺序运行。
      1. 仅TIV电动汽车
        1. 关闭Brightfield以获得1000个事件(用于补偿)。
        2. 开启Brightfield可以获取1000-5000个事件,以帮助初步确定门。
      2. 抗体单色对照(如上述步骤C5g所获取)
      3. 具有TIV和所有抗体的样品(亮域已打开)

数据分析

  1. Amnis IDEAS数据分析软件
    1. 电动汽车使用IDEAS(美国华盛顿州西雅图的阿姆尼斯)进行了量化。
    2. 使用IDEAS补偿矩阵向导创建实验补偿矩阵。
    3. 打开来自一个样本的原始数据文件,并应用补偿矩阵。
    4. 使用IDEAS软件中基于抗体和Tag-It Violet染色面板的逐步数据分析向导创建门控策略模板。图1说明了在流式细胞术数据分析中用于PTC识别的门控策略步骤。
      对于PTC识别,主要关注Tag-It Violet + / PL-VAP + / CD31- / CD144-事件,并为此目的开发了门控策略。 PL-VAP + / CD31- / CD144-的组合用于鉴定从肾周毛细血管释放的EV。


      图1.示意图,显示了用于PTC识别的流式细胞术门控策略。。步骤1:从收集到的所有事件中绘制出侧面散射(大小)和Tag-It紫罗兰色(EV)强度的散点图,绘制门(ROI) )对于Tag-It紫罗兰色阳性事件和SSC低;步骤2:EV +人群的PL-VAP抗体强度直方图,绘制PL-VAP阳性事件的门(ROI);步骤3:来自PL-VAP +人群的CD31和CD144散点图,对于PTC EV,最重要的门(ROI)是CD31- / CD144-,但是用于识别CD31- / CD144 +,CD31 +,CD144-和CD31 +的门(ROI) / CD144 +事件也会被绘制。

    5. EV使用以下流控策略进行测量(请参见图1)。
      1. 创建侧向散射(SSC)通道6与收集的所有事件的Tag-It Violet通道7的散点图。
        1. 绘制感兴趣的区域(ROI),以进行积极的Tag-it紫罗兰事件和低SSC
        2. 标记为Tag-It Violet +或EV +人群
      2. 创建PL-VAP通道的直方图。
        1. 得出正PL-VAP的ROI
        2. 标记为PL-VAP +
      3. 创建CD31与CD144的散点图
        1. 围绕负CD31和负CD144的事件绘制ROI。
        2. 标签为CD31- / CD144-
        3. 同时为CD31 + / CD144 +事件绘制投资回报率
    6. 具有所有ROI的门控策略在IDEAS中保存为模板。
      1. 保存的模板和补偿矩阵将应用于所有样本。
      2. 手动验证了3-5个样本的ROI,以验证ROI边界阈值
      3. 从每个样本中提取作为每个ROI中事件数的数据。
    7. 门控种群表示为Tag-It紫罗兰阳性事件的百分比。 (参见Sun et al。(2018),图2A和2C,以比较患者组之间的数据。)
      1. PL-VAP + / CD31- / CD144-事件
      2. PL-VAP + / CD31 +
      3. PL-VAP + / CD144 +
      4. PL-VAP + / CD31 + / CD144 +

  2. 使用JMP进行统计分析;请参阅Sun et al。(2018)进行数据分析。

菜谱

  1. EV缓冲液(pH 7.4)
    2 mol / L蔗糖(Sigma,BioXtra)
    500 mmol / L MES(西格玛)
    在4°C下稳定6个月。
  2. 5 mM Tag-It Violet TM (TIV)解决方案
    50μl无水DMSO
    一小瓶Tag-It Violet TM (122.25μg)
    在-20°C下可稳定1个月,但最佳做法是新鲜制作

致谢

该协议改编自Sun et al。(2018)。这项工作得到了美国国立卫生研究院(National Institutes of Health)资助号DK10081,DK104273,DK102325和DK120292的部分支持。

利益争夺

没有利益冲突。

伦理

这项研究已由梅奥诊所的机构审查委员会批准,并根据《赫尔辛基宣言》的道德原则进行。每位患者均获得知情的书面同意。

参考文献

  1. Aghajani Nargesi,A.,Lerman,L.O.和Eirin,A.(2017年)。 间充质干细胞来源的细胞外囊泡用于肾脏修复:现状和迫在眉睫的挑战。 干细胞研究 8(1):273。
  2. 卡普西利(M.),卡佩利(I。),G。Comai,G.Cianciolo和G.La Manna(2018)。 与中性粒细胞明胶酶相关的lipocalin作为肾移植后同种异体移植功能的生物标记:评估当前状态和未来的见解。 人工器官 42(1):8-14。
  3. Conley,S.M.,Shook,J.E.,Zhu,X.Y.,Eirin,A.,Jordan,K.L.,Woollard,J.R.,Isik,B.,Hickson,L.J.,Puranik,A.S.,和Lerman,L.O.(2019)。 代谢综合征可诱导猪间充质干细胞释放较小的细胞外囊泡。 细胞移植(未发布)。
  4. Hogan,MC,Manganelli,L.,Woollard,JR,Masyuk,AI,Masyuk,TV,Tammachote,R.,Huang,BQ,Leontovich,AA,Beito,TG,Madden,BJ,Charlesworth,MC,Torres,VE, LaRusso,NF,Harris,PC和Ward,CJ(2009年)。 PKD蛋白阳性外泌体样囊泡的特征。 J Am Soc肾上腺素 20(2):278-288。
  5. Kwon,S.H.,Woollard,J.R.,Saad,A.,Garovic,V.D.,Zand,L.,Jordan,K.L.,Textor,S.C.和Lerman,L.O.(2017年)。 肾血管性高血压患者尿中足细胞衍生的细胞外微泡升高。 移植 32(5):800-807。
  6. Mendt,M.,Kamerkar,S.,Sugimoto,H.,McAndrews,KM,Wu,CC,Gagea,M.,Yang,S.,Blanko,EVR,Peng,Q.,Ma,X.,Marszalek,JR ,Maitra,A.,Yee,C.,Rezvani,K.,Shpall,E.,LeBleu,VS,and Kalluri,R.(2018)胰腺癌的临床级外泌体的产生和测试。 JCI Insight 3(8):e99263。
  7. Santelli,A.,Sun,I.O.,Eirin,A.,Abumoawad,A.M.,Woollard,J.R.,Lerman,A.,Textor,Stephen C.,Puranik,A.S.和Lerman,L.O.(2019年)。 高血压患者的衰老肾细胞释放出尿液中的细胞外囊泡。 Journal美国心脏协会的报告 8(11):e012584。
  8. Sun,I. O.,Santelli,A.,Abumoawad,A.,Eirin,A.,Ferguson,C.M.,Woollard,J.R.,Lerman,A.,Textor,S.C.,Puranik,A.S.和Lerman,L.O.(2018)。 高血压患者的肾小管毛细血管丢失可通过尿道内皮微粒水平检测到。 高血压 72(5):1180-1188。
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引用:Woollard, J. R., Puranik, A., Jordan, K. L. and Lerman, L. O. (2019). Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine. Bio-protocol 9(21): e3420. DOI: 10.21769/BioProtoc.3420.
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