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Quantitative Analysis of Exosome Secretion Rates of Single Cells
单细胞外泌体分泌速率的定量分析   

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Abstract

To study the inhomogeneity within a cell population including exosomes properties such as exosome secretion rate of cells and surface markers carried by exosomes, we need to quantify and characterize those exosomes secreted by each individual cell. Here we develop a method to collect and analyze exosomes secreted by an array of single cells using antibody-modified glass slides that are position-registered to each single cell. After each collection, antibody-conjugated quantum dots are used to label exosomes to allow counting and analysis of exosome surface proteins. Detailed studies of exosome properties related to cell behaviors such as responses to drugs and stress at single cell resolution can be found in the publication (Chiu et al., 2016).

Keywords: Single cell(单细胞), Exosome(外来体), Single-cell assay(单细胞测定), Exosome secretion(外来体分泌), Exosome quantification(外来体定量), Single cell arrays(单细胞阵列), Single cell culture(单细胞培养)

Background

Exosomes have been found to play an essential role in tumorigenesis, cell-cell signaling, organotropic metastasis, drug resistance, and many crucial biological processes involving cell-cell communications. Most exosome isolation methods developed to date use ultracentrifugation at 100,000 x g (Théry et al., 2006) and require a large amount of samples. Combinations of microfluidics with immunological separation or physical trap have been reported (Liga et al., 2015) as simpler exosome isolation methods requiring a relatively small amount of sample. However, most microfluidic platforms have difficulties in integration of standard cell culture protocols, while cell culture in microfluidic environments can introduce new variables and unintended stresses to cells and change their behaviors and gene expressions. Above all, all existing approaches collect exosomes from cells without distinction, so it is extremely difficult to trace exosomes to the cells that secrete them. However, given the high diversity and inhomogeneity of biological samples, it is of great value to correlate the exosomes to the cell source. Furthermore, it is highly desirable to quantify the exosome analysis at a single cell level by finding the changes in exosome properties and secretion rates when cells are affected by stimuli, stresses and/or environmental changes. Here we provide a culture friendly, high-throughput, and versatile single-cell assay that enables quantitative analysis of exosomes secreted by individual cells.

Materials and Reagents

  1. 35 mm Petri dish (Corning, Falcon®, catalog number: 351008 )
  2. Cover glass (Ted Pella, catalog number: 260364 )
  3. Glass slides
  4. Silicon wafer (4” test grade) (University Wafer, catalog number: 452 )
  5. Acrylic (PMMA, 2 mm thick) (Sigma-Aldrich, catalog number: GF10188996 )
  6. 35 mm cell culture dish (Sigma-Aldrich, catalog number: D7804 )
  7. Parafilm (Sigma-Aldrich, catalog number: P7793 )
  8. Aluminum foil
  9. 0.22 µm filter
  10. Cells
    Note: Our protocol can be used for both adherent and non-adherent cells. For examples, MCF7, MB-MDA-231, MCF10A, Neuronspheres, etc.
  11. Polydimethylsiloxane (PDMS) (Dow Corning, catalog number: SYLGARD® 184 Silicone Elastomer Kit )
  12. Pure ethanol (Decon Labs, catalog number: V1001 )
  13. (3-mercaptopropyl)trimethoxysilane (MPS) (Gelest, catalog number: 4420-74-0 )
  14. Sulfo-GMBS, N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 22324 )
  15. Phosphate-buffered salines (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  16. Monoclonal anti-human CD63 antibody (Ancell, catalog number: 215-820 )
  17. Bovine serum albumin (BSA power) (Sigma-Aldrich, catalog number: A8531 )
  18. Paraformaldehyde solution (4% in PBS) (Affymetrix, catalog number: 19943 1 LT )
  19. Biotinylated anti-CD63 (Ancell, catalog number: 215-030 )
  20. Qdot® incubation buffer (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: Q20001MP )
  21. Quantum dots (Qdot® 545 ITKTM) streptavidin conjugate (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: Q10091MP )
  22. Tris buffered saline with Tween® 20 (TBST-10x) (Cell Signaling Technology, catalog number: 9997 )
  23. DI water
  24. Blocking buffer (see Recipes)
  25. Blocking buffer for Qdot (see Recipes)
  26. 1x TBST (see Recipes)

Equipment

  1. CNC (Computer Numeric Control) micro milling machine (Minitech Machinery, model: Mini/Mill/1 )
  2. Disco automatic dicing saw 3220 (Nano3, model: 3220)
  3. Plasma Etch system (for cleaning) (Plasma Etch, model: PE-100 )
  4. Soda Lime Silica glass Petri dish (Corning, catalog number: 70165-152 )
  5. Standard biosafety cabinet
  6. Shaker (VWR, model: VWR Mini shaker )
  7. Oven
  8. 1 ml pipet
  9. Microscope (KEYENCE, model: BZ-9000 , similar to BZ-X700 )
  10. Centrifuge (Thermo Fisher Scientific, model: CL2 )
  11. Tweezers
  12. Cell counter

Notes

  1. Equipment 1-3 can be found in core facility such as micro/nano fab. Please see Nano3 facility of UCSD as an example. Equipment 1 can also be found in most machine shop as a service.
  2. Centrifuge is chosen to fit 35 mm dishes. If you have a centrifuge that suit 6-well plate, 35 mm dishes can be replaced with a 6-well plate.

Procedure

  1. Overall procedure for Single-cell assay used for analyzing exosome secretion is shown in Figure 1. Details for preparing each material will be described in step 2.


    Figure 1. Workflow for single-cell assay used for analyzing exosome secretion. A and B. Loading single cells onto a culture dish with a cell loading chip that contains through hole arrays. C. The cell loading chip may be removed after cell attachment. D. A surface functionalized glass slide is placed in a CNC machined frame 100 µm above the cells. E. The glass slide with collected exosomes secreted by the corresponding cells. The captured exosomes are labeled with another biotinylated antibody and streptavidin-conjugated quantum dots to become visible under fluorescent microscope.

  2. Prepare a cell loading chip that can fit into a 35 mm dish. A chip size between 5 x 5 mm2 and 12 x 12 mm2 is recommended. A cell loading chip made of PDMS is also recommended (Chiu et al., 2016).
  3. CNC machined fixture
    1. Design a fixture with an outer diameter fitted to a 35 mm dish. Inner square of the fixture is 50 μm greater (on each side) than the cover glass used for exosome collection (Figure 2).


      Figure 2. A machined PMMA that fit a 35 mm dish and exosome collection cover glass

    2. Use CNC system to machine PMMA
      1. For adherent cell or cell loaded directly onto the dish, four diced silicon slides (100 μm x 3 mm) are attached (Note: we use PDMS to attach) to the inner corners as spacers between the cells and the collection glass (Figure 3).
      2. For non-adherent cell, the machined PMMA can be used after wash.
    3. Clean the machined PMMA using 75% ethanol and UV for 15 min.
      Note: We use standard biosafety cabinet UV lamp.


      Figure 3. Fixture with 4 100 µm spacers to keep the distance between the cover glass and cells

  4. Antibody immobilization on cover glass (this step will take about 4-6 h)
    1. Dice cover glass into 7 x 7 mm size.
    2. Using the dicing machine to cut ~30 μm depth fiducial on one side of cover glass. The design of fiducial is shown in Figure 4.


      Figure 4. Cover glass with 30 μm depth fiducial

    3. Clean the cover glass with O2 plasma (Plasma Etch System, PE100) at 200 W for 1.5 min on each side of cover glass.
    4. Silanization
      1. Prepare a sterile 35 mm glass dish.
        Note: For silanization we used a glass dish rather than plastic dish.
      2. Add 3 ml ethanol into the dish.
      3. Add 120 μl (3-mercaptopropyl)trimethoxysilane (MPS) into the dish (to get 4% v/v MPS) and mix well.
      4. Immerse cover glass in 4% MPS solution for 30 min on a shaker at 200 rpm.
        Note: Flat surface without fiducial facing up.
      5. Prepare a new 35 mm Petri dish with 3 ml ethanol.
      6. Transfer glass slides into the new dish and shake for 5 min at 200 rpm.
      7. Repeat steps 3d.v and 3d.vi two additional times.
      8. Transfer the glass slides into a new glass dish.
      9. Put the glass slides into a 100 °C oven and dry the slides for 30 min (until fully dry).
        Note: Freshly used. Go to next step immediately.
    5. Sulfo-GMBS coating
      1. Weigh 0.764 mg Sulfo-GMBS.
      2. Dissolve Sulfo-GMBS into 2 ml PBS to get 1 mM sulfo-GMBS solution in a 35 mm glass dish.
      3. Immerse the cover glass in the solution.
      4. Put the glass dish on the shaker at 200 rpm and incubate for 10 min and another 30 min without shaking.
      5. Rinse with PBS (3 ml) for 5 min 3 times on the shaker at 200 rpm.
        Note: Here we use plastic dish for PBS washing.
    6. Anti-CD63 Ab immobilization
      1. Prepare a 35 mm Petri dish.
      2. Add 2 ml PBS into the dish and shake gently to wet the whole surface.
      3. Add 5 μg (5 μl) anti-CD63 Ab into the dish (~0.05 μM).
      4. Use 1 ml pipet to mix the solution 20 times.
      5. Immerse the cover glass into the anti-CD63 Ab solution.
      6. Incubate the dish at 4 °C for 2 h.
      7. Rinse the dish with PBS (3 ml) for 5 min 3 times on the shaker at 200 rpm.
    7. Block Non-reacted GMBS
      1. Pipet 3 ml 5% bovine serum albumin (BSA) solution into a sterile 35 mm dish.
      2. Immerse the glass into BSA solution for 30 min at 4 °C.
    8. Storage
      1. Immerse the treated cover glass in PBS at 4 °C.
        Notes: 
        1) There is no need to wash with PBS after BSA blocking.
        2) The treated cover glass can be stored for at least 2 months. (We have never tried longer than this duration.)
  5. Single-cell loading
    1. Adhere cell loading array (through hole PDMS for adherent cell or PDMS wells for non-adherent cells) at the center of 35 mm cell culture dish. Figure 5 is an example of through hole PDMS adhere to a 35 mm dish.


      Figure 5. Through hole PDMS adhere to a 35 mm dish

    2. Place the CNC machined fixture on top of cell loading array.
      Note: For non-adherent cells, loading the cells after placing the fixture; for adherent cells, placing the fixture after loading and peel off the PDMS mesh.
      1. If the fixture is designed to fit the inner diameter of 35 mm dish, it can be placed directly on top of PDMS.
      2. If the fixture is not designed to fit the inner diameter of 35 mm dish, one would need to use some uncured PDMS to seal the side of fixture to the cell loading array. Make sure PDMS is fully cured before use.
    3. Use O2 plasma to treat the whole dish (with cap open) for 30 sec at 200 W. This will make PDMS more hydrophilic.
    4. Sterilize the whole dish under UV (15 W) for 15 min.
    5. Dispense 2-3 ml culture media into the dish and store in 37 °C overnight (at least 4-6 h depends on the well size). This step will help remove small bubbles trapped in PDMS wells, and ensure no bubbles after ramping up temperature.
    6. Before harvesting the cells, check under microscope and make sure there are no bubbles in the PDMS wells.
    7. Harvest the cell based on normal procedure.
    8. Dilute the cells with culture media for a suitable density. For a 50 x 50 array of loading wells with size of 40-50 μm and 250 μm separation (center to center), about 50,000-100,000 cells per ml will give good number of single cells.
    9. Pipet diluted cells into the dish, and homogenize the cells by pipetting the diluted cells a few times.
    10. Seal the dish with Parafilm.
    11. Put the dish in the centrifuge and use tape of cap to secure the dish. Two dishes each time will help to balance centrifuge easily (Video 1).

      Video 1. Loading cells by centrifuge

    12. Centrifuge for 1 min at 140 x g.
    13. After centrifuging, check the loading condition under microscope (2x or 4x of objective lens). You will see single/double cells cover more than 75% of wells. If loading efficiency is too low, one can repeat steps 4i-4l.
    14. Clear unwanted cell
      1. For adherent cells, let the cells settle for 6-8 h before removing the PDMS mesh. After removal, the unwanted cells will be taken away with the PDMS mesh. Wash the whole plate with media for 3 times.
      2. For non-adherent cells, after centrifuge, tilt the dish to 45-60 degree and use pipet to flush the surface. Repeat this step with clean media for 2-3 times.
  6. Exosome collection
    Note: Steps 6, 7, and 8 will take about 10 h including 3 h of exosome collection. We recommend finish 6-8 in one day without interruption.
    1. To start the collection process, aspirate the media in the dish and rinse with exosome free media or PBS.
    2. Dispense exosome free media into the dish.
    3. Carefully take out the treated cover glass from step 3 (Video 2).

      Video 2. Place the treated cover glass on top of the cells loading array

    4. Flip the cover glass. Let the flat surface without fiducial face down. Put the glass slide in the CNC machined PMMA fixture (Video 2).
    5. Put the cells back to the incubator and wait for 1-3 h based on the experimental plan.
  7. Image sample and exosome fixation
    1. After the exosome collection, capture whole dish images with microscope. Use the fiducial to find the focal plane.
    2. Prepare a 35 mm Petri dish filled with 2 ml of 4% paraformaldehyde in PBS.
    3. Use tweezers to take out the cover glass.
    4. Flip the cover glass and let the flat side (without fiducial) face up, and immerse the slide immediately to the paraformaldehyde for 30 min at room temperature.
      Note: Don’t forget to flip the cover glass.
  8. Label Qdots
    1. After exosome fixation, wash with 1x PBS for 3 times, each time 5 min at 200 rpm.
    2. Blocking
      1. Immerse the cover glass in 5% BSA for 2 h at room temperature.
      2. After blocking, wash with 1x PBS for 3 times, each time 5 min at 200 rpm.
    3. Label Biotinylated anti-CD63 Ab or other antibody
      1. Prepare a 35 mm Petri dish.
      2. Create a solution of 800 μl of PBS, 200 μl of 5% BSA, and 2.5 μl of Biotinylated anti-CD63 Ab.
      3. Immerse the cover glass in the solution for 2 h at 4 °C.
      4. Wash with 1x PBS for 3 times, each time 5 min at 200 rpm.
    4. Blocking
      1. Prepare a 5% BSA in Qdots incubation buffer.
      2. Dispense 5% BSA in Qdots incubation buffer into a 35 mm Petri dish.
        Note: It is recommended to use 500 μl for a 7 x 7 mm-12 x 12 mm glass slides to save the cost of Qdots. Add the blocking buffer from the side of dish till the buffer covers the glass.
      3. Immerse glass slide in the buffer for 2 h at room temperature.
    5. Qdots labeling
      1. Mix well 1 ml Qdots incubation buffer and 10 μl Qdots to get 10 nM Qdots solution.
      2. Immerse the cover glass in the solution for 1 h at room temperature. Cover the dish with aluminum foil.
        Note: No washing process before this step.
    6. Washing
      1. Preheat 1x TBST buffer and DI water at 50 °C for 30 min before Qdots labeling finishing.
      2. Add 2 ml 50 °C 1x TBST into a new dish, and transfer the cover glass to the dish with 1x TBST solution.
      3. Put the dish on the shaker and wash for 5 min at 200 rpm.
      4. Repeat steps 7f.ii-7f.iii for three times.
      5. Add 2 ml 50 °C deionized water (DI water) into another dish, and transfer the glass slide into it.
      6. Put the dish on the shaker and wash for 5 min at 200 rpm.
      7. Repeat steps 7f.v-7f.vi for three times.
    7. Dehydration
      1. Dispense 2 ml of 30% ethanol, 50% ethanol, 75% ethanol, 90% ethanol, and two portions of 100% ethanol in each clean dish.
        Note: The serial graded ethanol solutions are prepared with sterilized ddH2O.
      2. Immerse the washed sample in 30% ethanol for 30 sec, 50% ethanol for 30 sec, 75% ethanol for 30 sec, 90% ethanol for 30 sec, and 2 dishes of 100% ethanol for 30 sec.
      3. Transfer the glass on a clean surface and let it air dry. Cover with aluminum foil during drying.
  9. Imaging and counting
    1. Register fiducial on exosome collected glass slide relative to the position of cell array.
      1. Open the image taken at step 6 that contains fiducial, e.g., cross pattern.
      2. Display the image on computer screen using 4x microscope objective lens.
      3. Cover the computer screen with a piece of transparency (Figure 6).


        Figure 6. Transparency with labeled cell positions (red dots) and fiducial (cross pattern)

      4. Use a black marker to trace the edge of fiducial on the transparency.
      5. Use a red marker to draw the cell sites on the transparency.
      6. One transparency can cover ~5 x 6 mm2 area on glass. Use another transparency on another cross fiducial position if the size of cell array is greater than the field of view of 4x objective.
    2. Place glass slide under an inverted microscope with the exosome side facing down (i.e., facing the objective lens of microscope). We use double-sided tape to adhesive the edge of the cover glass on a glass slide.
      Note: Double-sided tape will contribute a lot fluorescent signal. Do not overlap the tape with the sample area.
    3. Register exosomes collection sites/cell loading sites
      1. At this step, we will register exosome collection sites on the glass slide to the center of the field of view initially using 2x/4x objective lens and then 100x objective lens to count the quantum dots that label the exosomes.
      2. Adhere the marked transparency on the screen (Figure 7A).
      3. Switch to 2x/4x objective lens or any low magnification.
      4. Move the sample stage until the fiducial on the glass slide is aligned with the fiducial drawn on the transparency (Figure 7B).
      5. Mark center position on the transparency. This will become the center field under 100x objective.
      6. Choose one cell loading site (Cell-1). Adjust the sample stage to move the chosen cell loading site to the center position (marked as Center in the Figure 7C). Record micrometer readings of the sample stage at this position as position-1.


        Figure 7. Register the first cell loading site. A. Viewing the image of glass slide on the computer screen covered by the marked transparency. B. Move the glass slide to align the fiducial with the marked position on the transparency. C. Move the chosen cell loading site (Cell-1) to the center position of the transparency, which is in the center field of 100x objective.

      7. To register the rest of exosome collection sites, simply count how many spots (red circles) the chosen site is away from the first cell (Cell-1). Under 2x/4x objective lens, move the micrometers of the sample stage according to the red circles on the transparency. This will move the exosome collection site to the center of image (Figure 8).
      8. Register all the exosome collection sites of interest by repeating step 9c.vii.


        Figure 8. Register the rest of cell positions

    4. Imaging
      1. Switch to 100x objective lens
      2. Apply real time haze reduction and random noise cancellation if applicable. This will help to identify Qdot signals from the noise signals generated due to long exposure time. Figure 9A shows an example of an image after haze reduction.
      3. Increase exposure time to 1.5-2.5 sec. It is easier to distinguish Qdots from random noise while using long exposure time. Although you may see both on the monitor, Qdots are always brighter than the noise.
      4. Adjust the focus slowly to see the Qdot signals.
      5. Optimize the focus carefully for the highest Qdot image contrast.
      6. Take image with a long exposure time, e.g., 2.5 sec is recommended.
    5. Qdots counting
      1. Black balance. Select an area and adjust the black balance until the dimmer Qdots show on the screen. Figure 9 shows an example noise reduction after black balance.
        Note: We suggest showing the image as original size while adjusting black balance. Some Qdots may not be visible due to the setting or capability of the computer screen.
      2. Use cell counter to count the illuminated dots. Carefully set the threshold.
      3. Repeat steps 8f.i and 8f.ii to count all the images.


        Figure 9. An example of Qdots image. A. Image after haze reduction and random noise cancellation. Red rectangular area shows background noise under very long exposure time. Yellow circles are examples of Qdots with different intensities. B. Image after black balance.

Data analysis


Where,
R: Single cell exosome secretion rate,
N: Number of Q-dots in the exosome collection slide area corresponding to the cell position,
Nn: Noise level,
C: Cell number in one site,
T: Exosome collection time.
We only imaged the sites that contain a single cell initially. If the cell divided during the experiment, we assumed the same exosome secretion rate for all the cells in the site.

Notes

  1. Washing: We use clean 35 mm Petri dish at every washing step.
  2. At step 8c, different antibodies can be chose to target the exosomes depending on the purpose of study. Here we use anti-CD63 antibody just as an example.
  3. At step 8e Imaging, if it is hard to find Qdots plane, one can follow the steps here:
    1. Under lower magnification (2x or 4x), move the fiducial to the center of the view.
    2. Switch to 100x magnification. The fiducial should still be seen within the field of view. If not, adjust the sample stage till the fiducial pattern shows in the field of view. As shown in Figure 10, try to focus on plane-1.
    3. Keep lowering the lens (i.e., bringing the lens away from the glass slide) until the lowest plane of fiducial is in focus marked as plane-2 in Figure 10. Now one can be sure the microscope is imaging a z-position between the top surface and the bottom surface of the glass slide.
    4. Keep lowering the lens, the Qdots and exosomes should be at the plane-3 shown in Figure 8.


      Figure 10. Find focal plane of exosomes

Recipes

  1. Blocking buffer
    1. Dissolve 0.5 g BSA into 25 ml PBS
    2. Filter the solution with 0.22 µm filter
    3. Store the 5% BSA buffer at 4 °C
  2. Blocking buffer for Qdot
    1. Dissolve 0.5 g BSA into 25 ml Qdots buffer
    2. Filter the solution with 0.22 µm filter
    3. Store the 5% BSA Qdots buffer at 4 °C
  3. 1x TBST
    Diluted 10x TBST by DI water

Acknowledgments

We acknowledge the article of journal publication on Small 2016, 12 (27), page 3658-3666. This protocol is modified from the experimental section of this Small article. The authors acknowledge the technical support of the staff of the San Diego Nanotechnology Infrastructure (SDNI), which is part of the National Nanotechnology Coordinated Infrastructure (NNCI). Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number R21GM107977 and the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under Award Number R43EB021129. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Yu-Hwa Lo has an equity interest in Nanocellect, Inc., a company that may potentially benefit from the research results. He is a co-founder of the company and a member of the company’s Scientific and Advisory Board.

References

  1. Chiu, Y. J., Cai, W., Shih, Y. R., Lian, I. and Lo, Y. H. (2016). A single-cell assay for time lapse studies of exosome secretion and cell behaviors. Small 12(27): 3658-3666.
  2. Liga, A., Vliegenthart, A. D., Oosthuyzen, W., Dear, J. W. and Kersaudy-Kerhoas, M. (2015). Exosome isolation: a microfluidic road-map. Lab Chip 15(11): 2388-2394.
  3. Théry, C., Amigorena, S., Raposo, G. and Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3: Unit 3 22.

简介

为了研究细胞群体内的不均匀性,包括外来体特征,如外来体外分泌细胞分泌速度和外来体携带的表面标志物,我们需要量化和表征每个细胞分泌的那些外来体。在这里,我们开发收集和分析由单个细胞阵列分泌的外来体的方法,使用位置登记到每个单细胞的抗体修饰的玻片。每次收集后,使用抗体缀合的量子点来标记外来体以允许外来体表面蛋白的计数和分析。在出版物(Chiu等人,2016)中可以找到与细胞行为相关的外来物质性质的详细研究,例如对药物的反应和单细胞分辨率的压力。

背景 已经发现外来体在肿瘤发生,细胞信号传导,有机转移,耐药性以及涉及细胞通信的许多关键生物学过程中起重要作用。迄今为止开发的大多数外来体分离方法使用100,000xg(Théry等人,2006)的超速离心,并且需要大量的样品。已经报道了微流体与免疫学分离或物理陷阱的组合,作为需要相对少量样品的更简单的外来体分离方法(Liga等人,2015)。然而,大多数微流体平台在标准细胞培养方案的整合方面有困难,而微流体环境中的细胞培养可以向细胞引入新的变量和非预期的应激,并改变其行为和基因表达。最重要的是,所有现有的方法从细胞中收集外来体,无差别,因此将外来体追踪到分泌细胞的细胞是非常困难的。然而,鉴于生物样品的多样性和不均匀性,将外来体​​与细胞来源相关联是非常有价值的。此外,非常需要通过在细胞受到刺激,应激和/或环境变化的影响时发现外来体特性和分泌速率的变化来量化单个细胞水平的外来体分析。在这里,我们提供了一种文化友好,高通量和多功能单细胞测定法,可以对个别细胞分泌的外来体进行定量分析。

关键字:单细胞, 外来体, 单细胞测定, 外来体分泌, 外来体定量, 单细胞阵列, 单细胞培养

材料和试剂

  1. 35 mm培养皿(Corning,Falcon ®,目录号:351008)
  2. 盖玻璃(Ted Pella,目录号:260364)
  3. 玻璃幻灯片
  4. 硅晶片(4"测试级)(大学晶圆,目录号:452)
  5. 丙烯酸(PMMA,2mm厚)(Sigma-Aldrich,目录号:GF10188996)
  6. 35mm细胞培养皿(Sigma-Aldrich,目录号:D7804)
  7. 石蜡膜(Sigma-Aldrich,目录号:P7793)
  8. 铝箔
  9. 0.22μm过滤器
  10. 细胞
    注意:我们的协议可用于贴壁和非贴壁细胞。例如,MCF7,MB-MDA-231,MCF10A,神经球等。
  11. 聚二甲基硅氧烷(PDMS)(Dow Corning,目录号:SYLGARD 184硅氧烷弹性体试剂盒)
  12. 纯乙醇(Decon Labs,目录号:V1001)
  13. (3-巯基丙基)三甲氧基硅烷(MPS)(Gelest,目录号:4420-74-0)
  14. Sulfo-GMBS,N- [γ-马来酰亚胺丁酰氧基]磺基琥珀酰亚胺酯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:22324)
  15. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  16. 单克隆抗人CD63抗体(Ancell,目录号:215-820)
  17. 牛血清白蛋白(BSA功率)(Sigma-Aldrich,目录号:A8531)
  18. 多聚甲醛溶液(PBS中4%)(Affymetrix,目录号:19943 1 LT)
  19. 生物素化抗CD63(Ancell,目录号:215-030)
  20. Qdot ®孵育缓冲液(Thermo Fisher Scientific,Molecular Probes TM,目录号:Q20001MP)
  21. (Thermo Fisher Scientific,Molecular Probes ,目录号:Q10091MP)的量子点(Qdot) 545 ITK TM
  22. 具有Tween 20(TBST-10x)(Cell Signaling Technology,目录号:9997)的Tris缓冲盐水
  23. 去水
  24. 阻塞缓冲区(见配方)
  25. Qdot阻塞缓冲区(见配方)
  26. 1 TB TB(见配方)

设备

  1. CNC(电脑数控)微型铣床(Minitech Machinery,型号:Mini/Mill/1)
  2. 迪斯科自动切割锯3220(Nano3,型号:3220)
  3. 等离子体蚀刻系统(用于清洁)(Plasma Etch,型号:PE-100)
  4. 苏打粉二氧化硅玻璃培养皿(康宁,目录号:70165-152)
  5. 标准生物安全柜
  6. 振动筛(VWR,型号:VWR Mini振动筛)
  7. 烤箱
  8. 1毫升移液器
  9. 显微镜(KEYENCE,型号:BZ-9000,类似于BZ-X700)
  10. 离心机(Thermo Fisher Scientific,型号:CL2)
  11. 镊子
  12. 细胞计数器

  1. 设备1-3可以在核心设施中找到,如微/纳米晶圆厂。请参见UCSD的Nano3设施。设备1也可以在大多数机器商店中找到。
  2. 选择离心机适合35毫米的菜肴。如果您有一个适合6孔板的离心机,则可以用6孔板代替35毫米的料理。

程序

  1. 用于分析外来体分泌的单细胞测定的总体程序如图1所示。准备每种材料的详细信息将在步骤2中进行描述。


    图1.用于分析外来体分泌的单细胞测定的工作流程.A和B.将单细胞加载到含有通孔阵列的细胞加载芯片的培养皿中。细胞装载芯片可以在细胞附着后去除。 D.将表面官能化的玻璃载玻片放置在细胞上方100微米的CNC加工框架中。 E.由相应细胞分泌的收集的外来体的玻片。捕获的外来体用另一种生物素化抗体和链霉抗生物素蛋白结合的量子点标记,在荧光显微镜下变得可见。

  2. 准备一个可装入35毫米盘的电池加载芯片。推荐在5 x 5 mm 2和12 x 12 mm 2之间的芯片尺寸。还建议由PDMS制成的电池加载芯片(Chiu等人,2016)。
  3. 数控加工夹具
    1. 设计一个外径为35毫米盘的夹具。夹具的内部平方度比用于外来物质收集的覆盖玻璃(图2)更大(每边)为50μm(图2)。


      图2.适合35毫米盘和外来物收集盖玻璃的加工PMMA

    2. 使用数控系统加工PMMA
      1. 对于直接装载到盘上的贴壁细胞或细胞,连接四个切片硅片(100μm×3mm)(注意:我们使用PDMS附着)作为细胞与收集玻璃之间的间隔物(图3) 。
      2. 对于非贴壁电池,加工后的PMMA可以在洗涤后使用。
    3. 使用75%乙醇和UV清洁机加工的PMMA 15分钟。
      注意:我们使用标准的生物安全柜UV灯。


      图3.带有4 100μm间隔物的夹具,以保持盖玻璃和电池之间的距离

  4. 将抗体固定在盖玻片上(此步骤约需4-6小时)
    1. 骰子盖玻璃成7×7毫米尺寸。
    2. 使用切割机在盖玻璃一侧切割〜30微米深的基准。基准的设计如图4所示

      图4.具有30μm深度基准的盖玻璃

    3. 使用O 2等离子体(等离子体蚀刻系统,PE100)在玻璃杯的每一侧用200W清洁玻璃盖1.5分钟。
    4. 硅烷化
      1. 准备一个无菌的35毫米玻璃皿 注意:对于硅烷化,我们使用玻璃皿而不是塑料盘。
      2. 将3ml乙醇加入盘中
      3. 加入120μl(3-巯基丙基)三甲氧基硅烷(MPS)到培养皿中(得到4%v/v MPS)并充分混合。
      4. 在200rpm的摇动器下将玻璃盖在4%MPS溶液中浸泡30分钟。
        注意:平面没有基准面朝上。
      5. 准备一个新的35毫升培养皿与3毫升乙醇
      6. 将玻璃片转入新碟中,以200rpm摇动5分钟。
      7. 再次重复步骤3d.v和3d.vi两次。
      8. 将玻璃片转移到新的玻璃皿中。
      9. 将玻璃载玻片放入100°C的烘箱中,将载玻片干燥30分钟(直到完全干燥) 注意:新鲜使用。立即进入下一步。
    5. Sulfo-GMBS涂层
      1. 称量0.764 mg Sulfo-GMBS。
      2. 将Sulfo-GMBS溶解在2ml PBS中,以在35mm玻璃皿中得到1mM sulfo-GMBS溶液。
      3. 将玻璃罩浸入溶液中。
      4. 将玻璃皿放在振荡器上,转速为200转/分,孵育10分钟,再摇动30分钟
      5. 用PBS(3ml)冲洗5分钟,摇动器以200rpm冲洗3次 注意:在这里我们用塑料盘洗PBS。
    6. 抗CD63抗体固定
      1. 准备35毫米培养皿。
      2. 将2ml PBS加入盘中,轻轻摇匀,使整个表面湿润。
      3. 将5μg(5μl)抗CD63 Ab加入盘中(〜0.05μM)
      4. 使用1毫升移液器将溶液混合20次
      5. 将盖玻片浸入抗CD63 Ab溶液中。
      6. 在4℃下培养皿2小时。
      7. 用PBS(3毫升)冲洗培养皿5分钟3次,摇床上以200转/分。
    7. 阻断未反应的GMBS
      1. 将3ml 5%牛血清白蛋白(BSA)溶液吸入无菌的35mm培养皿中
      2. 在4℃下将玻璃浸入BSA溶液中30分钟。
    8. 存储
      1. 将处理后的玻璃杯浸入4℃的PBS中 注意:
        1)在BSA封锁后,无需用PBS清洗。
        2)被处理的护罩玻璃可以储存至少2个月。 (我们从未尝试过比这个持续时间更长的时间。)
  5. 单电池加载
    1. 在35毫米细胞培养皿的中心附着电极负载阵列(用于贴壁细胞的通孔PDMS或用于非贴壁细胞的PDMS孔)。图5是通孔PDMS粘附到35mm盘的示例。


      图5.通孔PDMS粘附到35 mm盘

    2. 将CNC加工的夹具放在电池加载阵列的顶部。
      注意:对于非贴壁细胞,放置固定装置后装入细胞;对于粘附细胞,在装载后放置固定装置并剥离PDMS网。
      1. 如果夹具设计成适合35毫米盘的内径,则可以将其直接放置在PDMS的顶部。
      2. 如果固定装置的设计不符合35 mm的内径,则需要使用一些未固化的PDMS将固定装置的侧面密封到电池装载阵列。使用前请确保PDMS完全固化。
    3. 使用O 2等离子体以200W处理整个盘(盖帽打开)30秒。这将使PDMS更亲水。
    4. 在紫外线(15 W)下灭菌整个餐具15分钟。
    5. 将2-3ml培养基分配到培养皿中并在37℃下保存过夜(至少4-6小时取决于孔的大小)。这一步将有助于清除PDMS井中捕获的小气泡,并在升温后确保没有气泡
    6. 在收集细胞之前,请在显微镜下检查,并确保PDMS孔中没有气泡
    7. 根据正常程序收获细胞。
    8. 用培养基稀释细胞以达到合适的密度。对于尺寸为40-50μm和250μm分离(中心到中心)的50×50荷载孔阵列,每毫升约50,000-100,000个细胞将产生良好数量的单细胞。
    9. 将稀释的细胞吸入培养皿中,并通过将稀释的细胞移液几次使细胞匀浆。
    10. 用Parafilm密封盘。
    11. 将盘放在离心机中,并使用盖子带固定盘子。每次两个菜肴都有助于平衡离心机(视频1)。

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    12. 在140 x x离心1分钟。
    13. 离心后,检查显微镜下的负载情况(物镜的2x或4x)。你会看到单/双胞细胞覆盖了75%以上的井。如果加载效率太低,可以重复步骤4i-4l。
    14. 清除不需要的单元格
      1. 对于贴壁细胞,让细胞沉淀6-8h,然后移除PDMS网。去除后,不需要的细胞将被剥离与PDMS网格。用介质洗涤整个板3次。
      2. 对于非贴壁细胞,离心后,将培养皿倾斜45-60度,并用移液管冲洗表面。用干净的介质重复此步骤2-3次。
  6. 外来物系集合
    注意:步骤6,7和8将需要约10小时,包括3小时的外来体收集。我们建议在一天内完成6-8,不间断。
    1. 开始收集过程中,吸入培养皿中的培养基并用无外来物质的培养基或PBS冲洗。
    2. 将外来体免费培养基分配到盘中
    3. 仔细取出第3步处理的护目镜(视频2)。

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      视频2.将经过处理的盖玻片放在加载阵列的单元格顶部
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    4. 翻盖盖玻璃。让平坦的表面没有基准面朝下。将玻璃滑块放入CNC加工的PMMA夹具(视频2)。
    5. 将细胞放回培养箱中,根据实验计划等待1-3小时。
  7. 图像样本和外来体固定
    1. 在外来体收集后,用显微镜拍摄整个菜肴图像。使用基准点找到焦平面。
    2. 准备一个装有2ml 4%多聚甲醛PBS的35 mm培养皿。
    3. 用镊子取出盖玻璃。
    4. 翻转盖玻片,让平面(无基准面)朝上,并将载玻片立即浸入多聚甲醛中室温30分钟。
      注意:不要忘了翻盖玻璃。
  8. 标签Qdots
    1. 在外来体固定后,用1x PBS洗涤3次,每次在200rpm下5分钟。
    2. 封锁
      1. 将盖玻片浸入5%BSA,室温2小时
      2. 封闭后,用1x PBS洗涤3次,每次在200rpm下5分钟。
    3. 标记生物素化抗CD63 Ab或其他抗体
      1. 准备35毫米培养皿。
      2. 创建800μlPBS,200μl5%BSA和2.5μl生物素化抗CD63 Ab的溶液。
      3. 将玻璃罩浸入溶液中4小时。
      4. 用1x PBS洗涤3次,每次在200rpm下5分钟。
    4. 封锁
      1. 在Qdot孵育缓冲液中制备5%BSA
      2. 将5%BSA的Qdot孵育缓冲液分配到35 mm培养皿中。
        注意:建议使用500μl7 x 7 mm-12 x 12 mm的幻灯片,以节省Qdot的费用。从碟子边添加封闭缓冲液,直到缓冲器覆盖玻璃。
      3. 将玻璃片在室温下浸入缓冲液中2小时。
    5. 质量标签
      1. 混合1 ml Qdots孵育缓冲液和10μlQdots,得到10 nM Qdot溶液
      2. 在室温下将盖玻片浸入溶液中1小时。用铝箔覆盖盘子。
        注意:在此步骤之前无洗涤过程。
    6. 洗涤
      1. 预热1x TBST缓冲液和去离子水在50℃下30分钟,然后再进行Qdot标记
      2. 加入2 ml 50°C 1x TBST放入新菜中,并用1 TB TB溶液将盖玻片转移到盘中。
      3. 将盘放在振荡器上,以200rpm冲洗5分钟。
      4. 重复步骤7f.ii-7f.iii三次。
      5. 加入2ml 50°C去离子水(DI水)到另一个盘中,并将玻璃片转移到其中
      6. 将盘放在振荡器上,以200rpm冲洗5分钟。
      7. 重复步骤7f.v-7f.vi三次。
    7. 脱水
      1. 在每个清洁盘中分配2ml 30%乙醇,50%乙醇,75%乙醇,90%乙醇和2份100%乙醇。
        注意:串联级分的乙醇溶液用无菌ddH 2 O制备。
      2. 将洗涤后的样品浸入30%乙醇中30秒,50%乙醇30秒,75%乙醇30秒,90%乙醇30秒,2次100%乙醇30秒。
      3. 将玻璃转移到干净的表面,让其风干。干燥时用铝箔盖住。
  9. 成像和计数
    1. 在外来体上注册基准相对于细胞阵列的位置收集的玻片。
      1. 打开在步骤6拍摄的包含基准,例如,交叉图案的图像。
      2. 使用4x显微镜物镜在电脑屏幕上显示图像。
      3. 用一块透明度盖住电脑屏幕(图6)

        图6.带有标记的单元格位置(红点)和基准(交叉图案)的透明度

      4. 使用黑色标记来追踪透明度上基准点的边缘。
      5. 使用红色标记在透明度上绘制单元格位置。
      6. 一个透明度可以覆盖玻璃上的〜5 x 6 mm 2 区域。如果单元阵列的大小大于4x目标的视野,则在另一个交叉基准位置使用另一个透明度。
    2. 将倒置显微镜放置在载玻片内,将外来体侧朝下(即,面向显微镜的物镜)朝下。我们使用双面胶带将玻璃盖的边缘粘贴在玻璃滑板上。
      注意:双面胶带会产生很多荧光信号。请勿将胶带与样品区重叠。
    3. 注册外来体收集站点/细胞装载位点
      1. 在此步骤中,我们将把玻片上的外来体收集位点注册到视野的中心,最初使用2x/4x物镜,然后用100x物镜计算标记外来体的量子点。
      2. 在屏幕上粘贴标记的透明度(图7A)。
      3. 切换到2x/4x物镜或任何低倍率。
      4. 移动样品台,直到玻璃滑块上的基准与玻璃上绘制的基准线对齐(图7B)。
      5. 标记中心位置的透明度。这将成为100x目标下的中心区域。
      6. 选择一个单元格加载站点(Cell-1)。调整样品台以将选定的单元格加载位置移动到中心位置(标记为图7C中的中心)。将该位置的样品台的千分尺读数记录为位置-1。


        图7.注册第一个单元格加载站点。 A.在标有透明度的计算机屏幕上查看玻片的图像。 B.移动玻璃滑块,将基准与透明胶片上的标记位置对齐。 C.将所选择的单元格加载位置(Cell-1)移动到透明度的中心位置,该中心位于100x目标的中心区域。

      7. 要注册其余的外来物种收集站点,只需计算所选择的位点离开第一个细胞的多少个斑点(红色圆圈)(Cell-1)。在2x/4x物镜下,根据透明度上的红色圆圈移动样品台的微米。这将使外来物种收集站点移动到图像中心(图8)。
      8. 通过重复步骤9c.vii注册所有外来物种收集站点。


        图8.注册其余的单元格位置

    4. 成像
      1. 切换到100x物镜
      2. 应用实时雾度减少和随机噪声消除(如果适用)。这将有助于从长曝光时间产生的噪声信号中识别Qdot信号。图9A示出了雾度降低之后的图像的示例。
      3. 将曝光时间增加到1.5-2.5秒。使用长时间曝光时间更容易区分Qdots和随机噪声。虽然您可能在显示器上看到,Qdots总是比噪音更亮。
      4. 缓慢调整对焦以查看Qdot信号。
      5. 仔细优化焦点,以获得最高的Qdot图像对比度。
      6. 拍摄长时间曝光的图像,例如,建议使用2.5秒。
    5. Qdots计数
      1. 黑平衡。选择一个区域并调整黑色天平,直到屏幕上显示调光器Qdots。图9显示黑平衡后的噪声降低示例。
        注意:建议在调整黑平衡时将图像显示为原始尺寸。由于计算机屏幕的设置或功能,某些Qdots可能不可见。
      2. 使用细胞计数器来计数照明点。仔细设置阈值。
      3. 重复步骤8f.i和8f.ii计数所有图像。


        图9. Qdots图像的示例。 A.雾度降低和随机噪声消除后的图像。红色矩形区域在非常长的曝光时间内显示背景噪音。黄色圆圈是具有不同强度的Q点的示例。黑色平衡后的图像。

数据分析


哪里,
R:单细胞外分泌细胞分泌率,
N:与细胞位置相对应的外来体收集载玻片区域中的Q点数,
N n :噪声级别,
C:一个站点中的单元号,
T:外来体收集时间。
我们最初只拍摄了包含单个单元格的网站。如果在实验过程中细胞分裂,我们假设该位点中所有细胞的外来体分泌速度相同。

笔记

  1. 洗涤:我们每个洗涤步骤都使用干净的35毫米培养皿。
  2. 在步骤8c,可以根据研究的目的选择不同的抗体靶向外来体。这里我们使用抗CD63抗体作为例子。
  3. 在步骤8e成像,如果很难找到Qdots飞机,可以按照以下步骤:
    1. 在较低的放大倍数(2x或4x)下,将基准点移动到视图的中心。
    2. 切换到100倍放大倍数。基准仍应在视野内看到。如果没有,请调整样品台,直到基准图案在视野中显示。如图10所示,尝试专注于平面1。
    3. 继续降低镜头(,使镜头远离玻璃片),直到基准面的最低平面在图10中被标记为平面-2。现在可以确定显微镜是在玻片的顶表面和底表面之间成像z位置。
    4. 持续降低镜片,Qdots和Exosomes应处于图8所示的平面-3处。


      图10.查找外来体的焦平面

食谱

  1. 阻塞缓冲区
    1. 将0.5g BSA溶解于25 ml PBS中
    2. 用0.22微米过滤器过滤溶液
    3. 将5%BSA缓冲液储存在4°C
  2. 阻止Qdot的缓冲区
    1. 将0.5 g BSA溶解于25 ml Qdot缓冲液中
    2. 用0.22微米过滤器过滤溶液
    3. 将5%BSA Qdot缓冲液储存在4°C
  3. 1x TBST
    用DI水稀释10倍TBST

致谢

我们承认2016年6月12日(27),第3658-3666页的期刊出版物。本协议从本小文章的实验部分进行修改。作者承认圣地亚哥纳米技术基础设施(SDNI)的工作人员的技术支持,该基金会是国家纳米技术协调基础设施(NNCI)的一部分。本出版物报道的研究得到了国家卫生研究院国家卫生研究所(NIGMS)的授权号R21GM107977和国家卫生研究院生物医学影像与生物工程国家研究所(NIBIB)的支持,获得了R43EB021129号。内容完全是作者的责任,并不一定代表美国国立卫生研究院的官方观点。余浩华拥有可能受益于研究成果的公司Nanocellect,Inc.的股权。他是该公司的共同创始人,也是该公司科学顾问委员会的成员。

参考文献

  1. Chiu,YJ,Cai,W.,Shih,YR,Lian,I. and Lo,YH(2016)。  用于时间推移研究外来体分泌物和细胞行为的单细胞测定。 12(27):3658-3666。
  2. Liga,A.,Vliegenthart,AD,Oosthuyzen,W.,Dear,JW and Kersaudy-Kerhoas,M。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/25940789"target ="_ blank">外来体分离:微流体路线图实验芯片 15(11):2388-2394。 >
  3. Théry,C.,Amigorena,S.,Raposo,G.和Clayton,A。(2006)。从细胞培养上清液和生物液体中分离和表征外来体。第3章:第3单元。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chiu, Y., Cai, W., Lee, T., Kraimer, J. and Lo, Y. (2017). Quantitative Analysis of Exosome Secretion Rates of Single Cells. Bio-protocol 7(4): e2143. DOI: 10.21769/BioProtoc.2143.
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