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Sep 2019

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Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument
使用微压痕仪对腺泡进行机械表征   

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Abstract

The linker of nucleoskeleton and cytoskeleton (LINC) complex is responsible for tethering the nucleus to the cytoskeleton, providing a pathway for the cell’s nucleus to sense mechanical signals from the environment. Recently, we explored the role of the LINC complex in the development of glandular epithelial acini, such as those found in kidneys, breasts, and other organs. Acini developed with disrupted LINC complexes exhibited a loss of structural integrity, including filling of the lumen structures. As part of our investigation, we performed a mechanical indentation assay of LINC disrupted and undisrupted MDCK II cells using a micro-indentation instrument mounted above a laser-scanning confocal microscope. Through a combination of force measurements acquired from the micro-indentation instrument and contact area measurements taken from fluorescence images, we determined the average contact pressure at which the acini structure ruptured. Here, we provide a detailed description of the design of the micro-indentation instrument, as well as the experimental steps developed to perform these bio-indentation measurements. Furthermore, we discuss the data analysis steps necessary to determine the rupture pressure of the acini structures. While this protocol is focused on the indentation of individual glandular acini, the methods presented here can be adapted to perform a variety of mechanical indentation experiments for both 2D and 3D biological systems.

Keywords: Bio-indentation (生物压痕), Micro-indentation (微压痕), Biomechanics (生物力学), Tissue mechanics (组织机械力), Nuclear mechanics (核力学), LINC Complex (LINC 复合体), Acinar development (腺泡发育)

Background

Bio-indentation measurements have emerged as means to measure the material properties of biological systems at length scales ranging from sub-cellular biopolymers to multi-cellular tissue structures. Nano-indentation instruments with micron sized probes and contact areas on the order of 10-100 nm2 have been used to measure the material properties of biopolymers and individual cells (Stolz et al., 2004; Sen et al., 2005; Strasser et al., 2007; Li et al., 2008). Similarly, micro-indentation instrument with millimeter sized indentation tips and contact areas on the order of 10-100 µm2 have been used to study the collective behavior and responses of cellular structures to external mechanical forces (Ahn et al., 2010; Levental et al., 2010; Schulze et al., 2017). Exploring cellular responses to mechanical queues from the micro-environment is crucial toward understanding how cells regulate growth, undergo differentiation or morphological changes, and express various genes (Vogel and Sheetz, 2006).

Transmission of mechanical signals from the cytoplasmic cytoskeleton to the nuclear envelope occurs through an assembly of proteins collectively referred to as the linker of nucleoskeleton and cytoskeleton (LINC) complex (Crisp et al., 2006). In addition to facilitating the mechanotransduction of signals to the nucleus, the LINC complex is responsible for tethering the nucleus to the cytoskeleton and regulating cytoplasmic filament organization (Crisp et al., 2006; Lei et al., 2009; Mellad et al., 2011; Tapley and Starr, 2013). Disruption of the LINC complex has been proposed as a potential pathway in the development of a variety of human diseases including cancerous development in glandular epithelia; a reduction in the expression of LINC complex proteins, including SUN1, SUN2, nesprin-2, and lamin A/C, has been observed in breast cancer tissues (Debnath and Brugge, 2005; Horn et al., 2013; Meinke et al., 2014; Matsumoto et al., 2015). However, the precise role that the loss of the LINC complex plays during malignant transformation is not well understood. Recently, we explored the effect of LINC complex disruption on the development and maintenance of higher-order cellular structures (Zhang et al., 2019). Development of glandular acini were hindered when the LINC complex was disrupted resulting in the loss of structural integrity and the filling of the lumens in MDCK II and MCF-10A acini. As part of our investigation into the role of the LINC complex in acinar development, we performed a bio-indentation assay of MDCK II acini to determine the average contact pressure required to induce rupture of individual acini structures. MDCK II acini with filled lumens resulting from the disruption of the LINC complex were found to require higher contact pressures to induce rupture compared to undisrupted MDCK II acini.

Here, we present the protocol for performing the mechanical indentation assay of individual MDCK II acini that was developed as part of our exploration into the role the LINC complex plays in acinar development. Through a combination of force measurements recorded using a micro-indentation instrument and fluorescence images captured through confocal microscopy, we measure the pressure necessary to mechanically rupture individual glandular acini. We discuss the design and operation of a micro-indentation instrument capable of applying vertical displacements on the order nanometers and measuring normal forces on the order of micro-Newtons. Additionally, we discuss the experimental protocol necessary to perform these indentation measurements and the data analysis necessary to determine the average contact pressure to rupture the acini structure. While this manuscript is focused on the indentation of individual acini, these methods can be adapted to perform a variety of mechanical indentation explorations of both 2D and 3D biological systems.

Materials and Reagents

The following materials and reagents are used as part of this bio-indentation protocol:

  1. 8-well chamber slide (Nunc Lab-Tek, catalog number:154534)

  2. 35 mm glass bottom Petri dish, 20 mm well size #0 thickness (Cell Vis: D35-20-0-N)

  3. Matrigel matrix basement membrane (Corning, catalog number: 35623)

  4. MDCK II cell line (gifted from Jennifer Lippincott-Schwartz)

  5. MCF-10A cell line (ATCC, CRL-10317; RRID: CVCL_0598)

  6. DMEM medium w/ 4.5 g/L glucose (Mediatech Cellgro, catalog number: 23-10-013-CM)

  7. Donor bovine serum (DBS) (Gibco, catalog number: 16030074)

  8. Ethylenediaminetetraacetic acid (EDTA) (Corning, catalog number: 46034CI)

  9. Sodium orthovanadate (Na3VO4) (Sigma Aldrich, catalog number: S6508)

  10. Sodium fluoride (NaF) (Sigma Aldrich, catalog number: 67414)

  11. Calcein AM (Invitrogen, catalog number: C3100MP)

  12. 0.25% trypsin (Corning, catalog number: 25053CI)

  13. Poly-L-lysine (Sigma Aldrich, catalog number: P4832)

  14. Phosphate buffered saline (PBS) solution 1x (Fisher Scientific, catalog number: BP243820)

  15. F-127 pluronic (Sigma Aldrich, catalog number: P2443)

  16. Millipore water

  17. MDCK cell growth medium (see Recipes)

  18. MDCK acini indentation medium (see Recipes)

  19. Matrigel wash solution (see Recipes)

  20. Acini dye medium (see Recipes)

  21. F-127 pluronic solution (see Recipes)

Equipment

The following equipment is used as part of this bio-indentation protocol:

  1. Micro-indentation Instrument:

    1. Sapphire indentation tip (Edmund Optics, catalog number: 48-430)

    2. Capacitance load cell

      1. Double-leaf cantilever beam (custom-built; spring stiffness k = 49.94 N/m)

      2. Capacitive probe (Lion Precision, model: C5R-0.8)

      3. Capacitive sensor (Lion Precision, model: CPL290)

    3. Nylon M6 threaded rod (McMaster-Carr, catalog number: 98882A249)

    4. Nylon M6 hexagonal nut (McMaster-Carr, catalog number: 93800A600)

    5. Aluminum mount for cantilever beam (custom-built)

    6. Vertical precision Z-positioner (Physik Instrumente, model: P-622.ZCL PIHera)

    7. Aluminum mounting adapter (custom-built)

    8. 25 mm compact XY linear translational stage (Thor Lab, model: LX20)

    9. Aluminum mounting flange (custom-built)

    10. Super glue gel control (Locktite)

  2. Nikon Eclipse Ti2-E Inverted Confocal Microscope:

    1. Nikon Ti2-Eclipse inverted microscope (Nikon)

    2. Nikon C2+ confocal scan head (Nikon)

    3. C2-DU3 detector unit (Nikon)

    4. DAPI filter cube 438/24 (Nikon)

    5. FITC filter cube 525/50 (Nikon)

    6. 561 long pass filter (Nikon, model: 561LP)

    7. Reflectance filter cube (Nikon, model: A1-C2 C165305)

    8. LU-N4 laser unit (Nikon)

    9. 4x microscope objective (Nikon, model: MRD00045)

    10. 10x microscope objective (Nikon, model: MRD00101)

    11. 20x microscope objective (Nikon, model: MRD00205)

    12. Automated translational stage (Nikon, model: 761794)

    13. perfect focus (Nikon)

  3. Petri dish support plate:

    1. Aluminum Petri dish support plate (custom-built)

    2. Thermocouple (Omega)

    3. Power supply (Velleman, model: PS613U)

    4. Temperature controller (Omega, model: CN7800)

    5. Nichrome heating wire (Pelican Wire, model: 2130N80DGS)

  4. O2 plasma cleaner (Plasma Etch, model: PE-50)

  5. Cell incubator

  6. Cell centrifuge


Description of Indentation Instrument

Bio-indentation measurements of individual acini structures are performed using a micro-indentation instrument mounted above an inverted microscope (Figure 1A). The micro-indentation instrument consists of a hemispherical indentation tip, a precision load cell, a vertical Z-positioner, an XY translational stage, and a male dovetail mounting flange (Figure 1B). The indentation instrument is mounted into the condenser slot of the inverted microscope and secured using a hexagonal set screw (Figure 1C). Below, we describe the individual components of the micro-indentation instrument used to perform the indentation assay reported by Zhang et al., 2019 (A detailed CAD rendering of the indentation instrument can be found in the work presented by Schulze et al., 2017). Alternatively, there are several commercially available micro-indentation instruments, including the Bruker Hysitron Biosoft indenter, that provide comparable levels of performance.

  The bio-indentation measurements reported by Zhang et al. (2019) were performed using a micro-indentation instrument with a 1.6 mm radius of curvature hemispherical sapphire indentation tip. The load cell of the instrument consists of a double-leaf cantilever beam with a spring stiffness of k = 49.94 N/m, and a capacitive probe with a 100 µm detection range. The normal force of the indentation tip is calculated from the measured vertical deflection of the cantilever beam and the spring stiffness. Calibration of the bio-indentation instrument was performed by hanging weights of known mass from the double-leaf cantilever beam and measuring the resulting displacement using the capacitance probe. The spring stiffness was determined by fitting a line to the resulting force vs displacement curve. To provide an offset from the load cell and to allow for easy removal and cleaning, the indentation tip is attached to a ~15 mm long nylon threaded rod; the indentation tip is superglued to the tip of the mounting rod and a complementary mounting nut is superglued to the load cell. To control the vertical displacement of the indentation tip, the load cell is mounted to a PI piezoelectric translational z-stage capable of a 250 µm travel range with 1 nm resolution. The translational z-stage is subsequently mounted onto a 2-axis linear translational stage, allowing for alignment of the indentation tip in the XY plane. A D3N male dovetail mounting flange is fixed to the top of the XY translational stage to enable the bio-indentation instrument to be mounted into the condenser slot of an inverted microscope. The bio-indentation instrument is controlled through a custom-written Labview program. This program controls the vertical displacement of the piezoelectric stage while recording the signal from the capacitive sensor to calculate the vertical deflection of the double-leaf cantilever beam.




Figure 1. Micro-indentation instrument. A. Bio-indentation measurements are performed using a micro-indentation instrument mounted above an inverted microscope. B. The micro-indentation instrument consists of a hemispherical indentation tip, a load cell to measure the normal force, a vertical Z-positioner with nanometer resolution, a manual XY translational stage, and a dovetail mounting flange. C. The indentation instrument is mounted into the condenser slot of the inverted microscope and secured in place with a hexagonal set screw.

   The bio-indentation instrument is mounted on a Nikon Eclipse Ti2-E inverted microscope equipped with a C2+ confocal imaging system and a motorized translational stage. The microscope is configured to capture FITC fluorescence images using the 488 nm excitation laser and a 525/50 dichroic filter cube (Figure 2A). Additionally, confocal reflectance microscopy is used to align the indentation tip directly above the microscope objective prior to the indentation measurement. For reflectance imaging, the 488 nm laser is used as a light source and a reflectance filter cube is installed in the detector unit allowing all light to pass through to the detector channel (Figure 2B).




Figure 2. Microscope optical configurations. The Nikon Eclipse Ti2-E Confocal microscope is configured to allow for either (A) fluorescence imaging of the acini structure during the indentation measurement, or (B) reflectance microscopy during the instrumental set-up to determine the center of the indentation tip.


  Glass bottom Petri dishes are secured during the indentation measurements using a custom-built Petri dish support plate that fits within the plate adapter of the Nikon Eclipse Ti-2 microscope (Figure 3). The glass bottom Petri dish is positioned over the objective hole in the center of the support plate (1) and is firmly secured using two mounting arms (2). The mounting arms apply pressure to the Petri dish lid to mitigate movement during the indentation measurement. A hole is drilled in the lid of the Petri dish to provide an opening for the bio-indentation instrument. To regulate the temperature during the indentation measurement, the plate is equipped with a thermocouple (3) and nichrome heating wire (4) that are connected to an on-off temperature controller. The power voltage and current settings of the power supply are set to maintain a desired temperature of 37 °C while minimizing the frequency of on-off cycles.




Figure 3. Petri dish support plate. Glass bottom Petri dishes are secured during the indentation measurement using a custom-built support plate that fits into the plate adapter of the microscope’s translational stage. The temperature of the Petri dish is regulated using a temperature controller, a thermocouple, and a nichrome heating element.

Software

The following software is used as part of this bio-indentation protocol and data analysis process:

  1. Nikon NIS-Elements AR imaging software (Version 4.30.02)

  2. National Instruments LabView (Version 2014)

  3. FIJI ImageJ (https://imagej.net/Fiji) (Version 1.52g)

  4. Microsoft Excel (Office 365)

  5. OriginPro graphing software (version 8.5)

Procedure

  1. Cell Culture and Acini Growth

    Note: A complete description of cell culture protocols, cell line development, and growth of acini structures are reported by Zhang et al. (2019).

    1. Culture and maintain MDCK II cells following standard cell culture protocols for thawing, feeding, and passaging. Incubate cells at 37 °C in a humidified, 5% CO2 environment. Cell culture medium ingredients and their corresponding concentrations are provided in the Recipes section of this protocol.

    2. Induce acinar formation of MDCK II cells in an 8-well chamber slide one week prior to performing the mechanical indentation measurement.

      1. Coat an 8-well Nunc Lab-Tek chamber slide with Matrigel by spreading 40-45 µl of Matrigel in each well of the chamber slide. Incubate for at least 30 min at 37 °C or until the gels have completely solidified.

      2. Trypsinize MDCK II cells following standard cell culture procedures. Centrifuge the cells at 125 x g for 15 min and remove the supernatant. Resuspend the MDCK II cells into MDCK acini indentation medium. MDCK acini indentation medium ingredients and their corresponding concentrations are provided in the Recipes section of this protocol.

      3. Seed the MDCK II cells into Matrigel coated 8-well chamber slide at a seeding density of ~5,000 cells/well with MDCK indentation medium. Incubate the chamber slides at 37 °C in a humidified 5% CO2 environment for approximately 7 days or until the MDCK cells have formed glandular acini structures.

    3. Transfer the acini structures into a glass bottom Petri dish for the indentation measurement.

      1. Coat the glass bottom Petri dish with 0.01 wt% poly-L-lysine. Place 200 µl of 0.01 wt% poly-L-lysine into glass bottom Petri dish and let sit for 1 hr. Remove the poly-L-lysine solution and rinse with 1x PBS solution. The poly-L-lysine should prevent any slippage of the acini structure during the indentation process.

      2. Remove the acini from the Matrigel growth matrix. Wash the acini in Matrigel coated 8-well chamber slide with a 1x cold PBS solution at 4 °C. Incubate the Matrigel coated 8-well chamber slide containing the grown acini in a 1x PBS solution supplemented with 5 mM EDTA, 1 mM NaVO4, and 1.5 mM NaF to remove the Matrigel growth matrix from the acini structure. Isolate the acini structures from the Matrigel support matrix by centrifuging at 4 °C for 15 min at 125 x g. Remove the supernatant and rinse the resulting pellet with a 1x PBS solution prepared at 4 °C. Centrifuge the acini structure again for 15 min at 125 x g and remove the supernatant. Resuspend the pellet into a 1x PBS solution at 4 °C and transfer the acini into poly-L-lysine coated glass bottom Petri dishes at a seeding density of ~1,000 acini/Petri dish. It is important to keep the acini density relatively low in the glass bottom Petri dishes to avoid contacting multiple acini structures during the indentation experiment.

    4. Dye the acini structures with calcein AM to check the viability of individual acini and enable fluorescence confocal microscopy of the acini during the indentation process. Replace the PBS solution with the appropriate cell growth medium supplemented with 1:2,000 parts calcein AM cell dye. Incubate the acini for 20 min at 37 °C in a humidified, 5% CO2 environment. Remove the medium and calcein AM solution and replace with fresh cell culture medium. Continue to incubate the acini at 37 °C in a humidified, 5% CO2 environment until it is time to perform the indentation experiment.


  2. Instrument Set-Up

    1. Mount the bio-indentation instrument and Petri dish support plate onto the confocal microscope. The bio-indentation instrument mounts onto the confocal using the condenser slot; slide the mounting flange of the bio-indentation instrument into the condenser slot and tighten the mounting screw until the bio-indentation instrument is securely mounted. Place the Petri dish support plate into the plate adapter on the confocal microscope’s translational stage. Connect the Petri dish support plate to the temperature controller and power supply unit, and adjust the power settings to maintain a temperature of 37 °C.

    2. Coat the indentation tip with F-127 pluronic to mitigate the effects of adhesion between the glass indentation tip and the acini structure. Remove the indentation tip from the double-leaf cantilever beam by unfastening the mounting screw from the mounting nut. Plasma clean the indentation tip using the O2 plasma cleaner for 1 min. Immediately after plasma cleaning, submerge the indentation tip into a 0.1 wt% F-127 pluronic solution for 10 min. Rinse the indentation tip with a 1x PBS solution before remounting the indentation tip onto the double-leaf cantilever beam.

    3. Adjust the height of the capacitance probe to ensure the double-leaf cantilever beam is within the detection range of the capacitance probe. The capacitance probe should be close enough to detect the cantilever beam but far enough away that the deflection of the cantilever beam during the indentation stays within the detection limits of the capacitance probe.

    4. Align the indentation tip over the center of the microscope objective. Beginning with the lowest magnitude objective, lower the indentation tip and adjust the objective focal point until the indentation tip is within focus. Using the XY linear translational stage, adjust the location of the indentation tip to align the center of the indentation tip with the center of the microscope’s field of view. Install the reflectance filter cube into the confocal microscope’s detector unit and load the reflectance imaging settings in the NIS-Elements software (Figure 2B). Adjust the focal plane of the confocal microscope to find the apex of the indentation tip. Continue to align the center of the indentation tip with the center of the microscope’s field of view.

    5. Repeat the alignment steps using progressively higher objective magnifications until the indentation tip is centered directly above the 20x microscope objective. It is recommended to use the 1.5x optical zoom feature during the indentation. Once the indentation is aligned over the microscope objective, remove the reflectance cube from the confocal microscope’s detector unit, re-install the 438/24 filter cube, and select the DAPI/FITC/TRITC imaging settings in the NIS-Elements software (Figure 2A). It is recommended at this time to check the indentation tip for any debris that may cause irregular surface contact between the indentation tip and the acini structure.

    6. Secure the glass bottom Petri dish on the confocal microscope’s translational stage. Place the glass bottom Petri dish containing the acini structures onto the Petri dish support plate. Secure the Petri dish to the support plate using the two mounting arms.

    7. Determine the location of the glass surface of the Petri dish and zero the indentation instrument’s force and displacement values. Navigate to a region in the center of the Petri dish in which there are no acini present in the microscope’s field of view. Initiate the indentation software and zero the force and displacement measurement. Drive the indentation tip 100 µm toward the surface of the Petri dish using the piezoelectric z-stage; at this time, the indentation tip should not be in contact with the surface and the force measurements should be fluctuating about 0 N. Manually lower the indentation instrument using the focal adjustment of the condenser stage until the indentation tip comes into contact with the glass surface as evident by a sudden and drastic increase in the measured force along with a decrease in the noise in the force measurement. Once contact with the surface has been made, slowly raise the indentation instrument using the focal adjustment of the condenser stage until the indentation tip is no longer in contact with the surface and the measured force returns to 0 N. Raise the indentation tip 100 µm using the piezoelectric z-stage. The indentation tip should now be located ~100 µm above the glass surface of the Petri dish – high enough to avoid contact with acini structures when moving the confocal microscope’s translational stage, but low enough to achieve contact during the indentation measurement.


  3. Mechanical Indentation of Acini Structure

    Mechanical indentations of glandular acini structures are performed to determine the average applied pressure at which the acini structures mechanically fail. The indentation tip is slowly brought down into contact with an individual acini structure at a fixed indentation rate while the applied force is recorded with simultaneous fluorescence imaging of the cells. As the indentation force exceeds the background level of noise in the measured force, the acini structures are observed to flatten out. Further increasing the applied force results in continued deformation of the acini until there is a structural collapse resulting in the formation of cellular blebs and/or the bursting of cells (Figure 4A). These rupture events are captured in the fluorescence images of the deforming acini enabling us to pinpoint the time at which the first rupture event occurs (Figure 4B). After the initial rupture, the indentation tip continues to push into the acini structure until the measured applied load reaches the specified maximum indentation force. Once the maximum indentation force is achieved, the indentation tip pulls off the structure at the designated indentation rate, returning to the starting z-position. To perform the mechanical indentation of an acini structure:

    1. Locate an isolated acini structure and align it in the center of microscope’s field of view, directly beneath the indentation tip. It is important that the acini structure is isolated within the microscope’s field of view to avoid contact with other acini structures. It is recommended to use brightfield imaging while locating the acini structures to avoid prolonged exposure of the acini structures to the confocal laser and photobleaching of the fluorescence dye.

        Misalignment of the acini structure can lead to irregular surface contact between the indentation tip and the acini resulting in improper force measurements as the indentation tip comes into contact with the glass substrate prior to the acini structure rupturing. A sudden, near vertical increase in the force vs displacement curve is a strong indicator that the acini structure is not properly aligned with the indentation tip.

    2. Navigate the focal plane of the confocal microscope to the desired imaging plane for the indentation measurement. It is recommended to choose a focal plane that is close to the bottom of the acini structure to fully capture the deformation during the indentation process (Figure 4A); a focal plane chosen too high may result in the acini structure deforming out of the imaging plane prior to the rupture event occurring.

      Note: It is optional to perform a confocal z-scan at this time to capture the shape of the acini structure prior to the indentation measurement.

    3. Prepare the confocal microscope for the indentation measurement. Initiate perfect focus in the confocal software and adjust the region of interest and the capture delay of the confocal software to achieve the fastest framerate possible. A higher framerate during the indentation measurement will result in an improved temporal resolution when determining the rupture pressure of the acini structure.

    4. Prepare the indentation instrument for the indentation measurement. Within the indentation instrument software, set the parameters for the indentation rate and maximum indentation force. We recommend using an indentation rate of 0.1 µm/s and a maximum indentation force of 100 µN. Faster indentation rates will reduce the temporal resolution of the fluorescence images captured throughout the measurement. A slower indentation rate will improve the temporal resolution when determining the rupture pressure but will increase the experimental time and laser exposure of the cells. The maximum indentation force should be set sufficiently high enough that the acini structure ruptures before the maximum force is reached. From our experience, a 100 µN maximum indentation load was sufficiently high enough to induce rupture of the acini; however, a higher maximum indentation force will not alter the results of the measurement.

    5. Simultaneously initiate the indentation measurement of the acini structure and the imaging time-lapse on the confocal microscope. Record any discrepancies in start times of the indentation measurement and imaging time-lapse.

Data analysis

Once the indentation has been completed, the rupture pressure of the acini structure is determined from the normal contact force measured by the indentation instrument and the contact area measured from the fluorescence images.

  1. Determine the time at which the acini structure first ruptures using the time-lapse images captured using the confocal microscope (Figure 4B). Correct the time for any discrepancies between the start times of the indentation measurement and the confocal time-lapse.

  2. Measure the contact area of the acini structure at moment in which the rupture event is first observed (Figure 4B). This can be accomplished using either the NIS Elements software or FIJI ImageJ. The contact area can be measured by drawing the best-fit circle around the acini structure at the moment of rupture.

  3. Determine the normal contact force experience by the acini structure at the time of rupture. This step can be accomplished using either Microsoft Excel or OriginLabs graphing software. Load the force vs time data into the graphing software and record the indentation force at the previously determined time of rupture.

  4. Calculate the rupture pressure of the acini structure from the measured contact area and the measured normal contact force. The average contact pressure for Hertzian contact of a sphere is given as <P> = F/A, where F is the measured normal force from the indentation instrument and A is the measured contact area from the confocal images.



    Figure 4. Data analysis from micro-indentation measurements. A. Schematic illustration of the bio-indentation experiment. The imaging plane is chosen close to the bottom of the acini structure to capture the full deformation during the indentation measurement. B. Fluorescence images captured through confocal microscopy show the deformation of the acini structure throughout the indentation. As the force is increased, the formation of cellular blebs and/or the bursting of cells can be observed between subsequent frames indicating the rupturing of the acini structure. In the presented example, we observe the rupture of the acini at t = 812 s as evident by rupture of a cell and the release of the calcein dye into the surrounding media. The contact area is determined by manually fitting a circle to the acini structure at the instance in rupture is observed. C. The indentation force at the time of rupture is determined from the force measurements of the bio-indentation instrument. (µN: micro-Newton)


Discussion

Bio-indentation provides a means to explore both the material properties of biological materials, as well as the collective behavior of cellular systems to externally applied forces. By coupling mechanical indentation with fluorescence images, we directly observe the deformation and rupture of individual glandular acini structures resulting from an externally applied mechanical force. This protocol was developed as part of our exploration into the role the LINC complex plays in the development and maintenance of glandular acini structures (Zhang et al., 2019). Acini formed from MDCK II cells with disrupted LINC complexes failed at higher applied pressures than those formed with intact LINC complexes. These results appear consistent with the observations of filled lumens in acini structures with disrupted LINC complexes. Furthermore, treating LINC-disrupted acini with Y-27632 resulted in the recovery of the rupture pressure to undisrupted levels.

  Although the protocol presented here is focused on performing bio-indentation measurements on individual acini structures, similar indentation protocols can be developed for exploring a variety of biological systems. For example, the elastic modulus and permeability of MDCK monolayers, and the minimum pressure necessary to rupture the nuclear envelope of 3T3 fibroblast cells have been investigated using similar micro-indentation protocols (Schulze et al., 2017; Halfmann et al., 2019). Furthermore, the effect of shear stresses and friction on biological systems can be studied with the addition of a second load cell to measure lateral forces (Urueña et al., 2018). Continued exploration into the response of biological systems to external forces and mechanical queues will be crucial for understanding collective cell behavior and developmental biology.

Recipes

Below are the recipes for the various solution used for culturing the MDCK cells and performing the bio-indentation protocol:

  1. MDCK cell growth medium

    DMEM medium

    4.5 g/L glucose

    10% v/v DBS

  2. MDCK acini indentation medium

    DMEM medium

    4.5 g/L glucose

    10% v/v donor bovine serum

    2 % v/v Matrigel matrix basement membrane

  3. Matrigel wash solution

    1x PBS solution

    5 mM EDTA

    1 mM Na3VO4

    1.5 mM NaF

  4. Acini dye medium

    cell growth medium

    Calcein AM (1:2,000)

  5. F-127 pluronic solution

    Water

    0.1 wt% F-127 pluronic

Acknowledgments

The indentation protocols presented here were developed as part of the research work of Zhang et al. (2019). This work was supported by the NIH (grant R01 EB014869) to T.P. Lele, and the NSF (grant DMR-1352043) to T.E. Angelini. The authors would like to acknowledge Dr. Kyle D. Schulze and Dr. W. Gregory Sawyer for their contribution in developing the micro-indentation instrument used in this protocol. The authors would also like to thank Dr. Jennifer Lippencott-Schwart for graciously donating the MDCK II cells.

Competing interests

The authors have no financial conflicts of interest to declare.

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简介

[摘要]的接头nucleoskeleton和细胞骨架(LINC)络合物负责核束缚到细胞骨架,提供用于细胞核从环境感测机械信号的通路。最近,我们探索了LINC复合物在腺上皮腺泡(例如在肾脏,乳房和其他器官中发现的腺泡)的发育中的作用。用破坏的LINC复合物开发的Acini表现出结构完整性的丧失,包括管腔结构的填充。作为研究的一部分,我们使用安装在激光扫描共聚焦显微镜上方的微型压痕仪对LINC破坏的和未破坏的MDCK II细胞进行了机械压痕测定。通过从微压痕仪获得的力测量值和从荧光图像获得的接触面积测量值的组合,我们确定了痤疮结构破裂的平均接触压力。在这里,我们提供了微压痕仪设计的详细描述,以及为执行这些生物压痕测量而开发的实验步骤。此外,我们讨论了确定痤疮结构破裂压力所需的数据分析步骤。尽管此协议专注于单个腺腺突的压痕,但此处介绍的方法可适用于针对2D和3D生物学系统执行各种机械压痕实验。

关键字:生物压痕,微压痕,生物力学,组织力学,核力学,LINC复合体,腺泡发育



[背景]生物压痕测试已成为手段长度尺度从亚细胞生物聚合物以多细胞组织结构来测量生物系统的材料性质。具有微米级探针和接触面积为10-100 nm 2的纳米压痕仪已用于测量生物聚合物和单个细胞的材料特性(Stolz等,2004; Sen等,2005; Strasser等)等人,2007; Li等人,2008)。同样,具有毫米大小的压痕尖端和接触面积为10-100 µm 2的微压痕仪已用于研究细胞结构对外部机械力的集体行为和响应(Ahn等人,2010; Levental等人等人,2010; Schulze等人,2017)。探索细胞对微环境中机械队列的反应,对于理解细胞如何调节生长,经历分化或形态变化以及表达各种基因至关重要(Vogel和Sheetz,2006)。

机械信号从细胞质细胞骨架到核被膜的传输通过蛋白质的组装发生,这些蛋白质被统称为核骨架和细胞骨架(LINC)复合物的连接子(Crisp et al。,2006)。除了促进信号向核的机械传递外,LINC复合体还负责将核束缚于细胞骨架并调节细胞质细丝的组织(Crisp等,2006 ;Lei等,2009;Mellad等,2011)。; Tapley和Starr,2013年)。有人提出破坏LINC复合体是多种人类疾病发展的潜在途径,包括腺上皮癌变。在乳腺癌组织中已观察到LINC复合蛋白(包括SUN1,SUN2,nesprin-2和层粘连蛋白A / C)的表达减少(Debnath和Brugge,2005年; Horn等人,2013年; Meinke等人。,2014 ; Matsumoto等,2015 )。如何过,的确切作用是在LINC复杂的戏剧的恶性转化过程中的损失还不是很清楚。最近,我们探索了LINC复合物破坏对高阶细胞结构的发育和维持的影响(Zhang等人,2019)。当LINC复合物被破坏,导致结构完整性丧失和MDCK II和MCF-10A腺泡内腔充盈时,腺腺腺泡的发育受到阻碍。作为我们对LINC复合体在腺泡发育中作用的研究的一部分,我们进行了MDCK II腺泡的生物压痕测定,以确定诱导各个腺泡结构破裂所需的平均接触压力。与未破坏的MDCK II腺泡相比,发现由于LIN C复合物的破坏而产生的内腔充满的MDCK II腺泡需要更高的接触压力才能引起破裂。

在这里,我们提出了对单个MDCK II腺泡进行机械压痕测定的方案,该方案是我们探索LINC复合物在腺泡发育中所起的作用的一部分。通过使用微压痕仪记录的力测量值和通过共聚焦显微镜捕获的荧光图像的组合,我们可以测量机械破裂单个腺腺泡所需的压力。我们讨论了一种微压痕仪的设计和操作,该压痕仪能够在纳米级上施加垂直位移,并能够测量微牛顿级上的法向力。此外,我们讨论了执行这些压痕测量所必需的实验规程以及确定平均接触压力以破坏acini结构所必需的数据分析。虽然此手稿着重于单个腺泡的压痕,但这些方法仍可适用于对2D和3D生物学系统进行各种机械压痕探索。



材料和试剂



以下材料和试剂用作该生物识别方案的一部分:

1. 8 -孔室载玻片(Nunc公司实验室-Tek公司,目录号:154534)

2. 35毫米玻璃底部培养皿,20毫米孔#0厚度(样品池可见:D35-20-0-N)

3.基质胶基质基底膜(Corning,目录号:35623)

4. MDCK II细胞系(赠自Jennifer Lippincott-Schwartz)

5. MCF-10A细胞系(ATCC,CRL-10317; RRID:CVCL_0598)

6.含4.5 g / L葡萄糖的DMEM培养基(Mediatech Cellgro ,目录号:23-10-013-CM)

7. D或牛血清(DBS)(Gibco,目录号:16030074)

8. ë thylenediaminetetraacetic乙酸(EDTA)(Corning公司,目录号:46034CI)

9.小号憎恨原钒(钠3 VO 4 )(Sigma Aldrich公司,目录号:S6508)

10.小号憎恨氟化物(氟化钠)(Sigma Aldrich公司,目录号:67414)

11. Ç alcein AM(Invitrogen公司,目录号:C3100MP)

12. 0.25%胰蛋白酶(Corning ,目录号:25053CI)

13. P -L-赖氨酸(Sigma Aldrich,目录号:P4832)

14. P hosphate缓冲盐水(PBS)溶液1×(Fisher Scientific公司,目录号:BP243820)

15. F-127普朗尼克(Sigma Aldrich,目录号:P2443)

16.密理博水

17. MDCK细胞生长培养基(请参阅食谱)

18. MDCK acini压痕介质(请参见食谱)

19.基质胶洗涤液(请参阅食谱)

20. Acini染料培养基(请参见配方)

21. F-127普朗尼克溶液(请参阅食谱)



设备



以下设备用作此生物识别协议的一部分:

中号ICRO压痕仪:
小号apphire压痕末端(埃德蒙光学,目录号:48-430)
Ç apacitance负载单元
d ouble叶悬臂梁(定制;弹簧刚度ķ = 49.94 N / M)
电容式探头(Lion Precision ,型号:C5R-0.8)
电容式电容传感器(Lion Precision ,型号:CPL290)
Ñ ylon M6螺纹杆(麦克马斯特-卡尔,目录号:98882A249)
Ñ ylon M6六角螺母(麦克马斯特-卡尔,目录号:93800A600)
悬臂梁的发光支架(定制)
V ertical高精度Z定位器(Physik公司INSTRUMENTE ,型号:P-622.ZCL PIHera )
一个luminum安装适配器(定制)
25 mm紧凑型XY线性平移台(Thor Lab,型号:LX20)
一个luminum安装法兰(定制)
小号UPER胶水凝胶控制(Locktite )
尼康Eclipse Ti2-E倒置共焦显微镜:
尼康Ti2-Eclipse倒置显微镜(Nikon)
尼康C2 +共焦扫描头(尼康)
C2-DU3检测器单元(尼康)
DAPI滤镜立方体438/24(Nikon)
FITC过滤器立方体525/50(Nikon)
561长通滤镜(尼康,型号561LP)
ř eflectance滤波器立方体(尼康,型号:A1-C2 C165305)
LU-N4激光单元(尼康)
4x显微镜物镜(尼康,型号:MRD00045)
10x显微镜物镜(尼康,型号:MRD00101)
20x显微镜物镜(尼康公司,型号:MRD00205)
甲utomated平移阶段(尼康,型号:761794)
完美对焦(尼康)
P ETRI盘支撑板:
甲luminum P ETRI盘支撑板(定制)
T型热电偶(欧米茄)
P奥尔供应(威尔曼,型号:PS613U)
Ť emperature控制器(欧米茄,型号:CN7800)
N ichrome加热丝(Pelican Wire,型号:2130N80DGS)
O 2等离子清洁剂(等离子蚀刻,型号:PE-50)
Ç ELL孵化器
Ç ELL离心机


压痕仪说明

使用安装在倒置显微镜上方的微压痕仪(图1A)对单个棘突结构进行生物压痕测量。微压痕仪由一个半球形压痕头,一个精密测力传感器,一个垂直Z形定位器,一个XY平移台和一个凸形燕尾安装法兰组成(图1B)。压痕仪安装在倒置显微镜的聚光镜槽中,并使用六角定位螺钉固定(图1C)。下面,我们描述了用于执行由Zhang等人报道的压痕测定的微压痕仪器的各个组件。,2019(压痕仪的详细CAD渲染可以在Schulze et al。,2017提出的工作中找到)。或者,有几种可商购的微压痕仪,包括Bruker Hysitron Biosoft压痕仪,它们可提供相当水平的性能。

张等人报道的生物压痕测量。(2019)使用具有1.6 mm曲率半径半球形蓝宝石压痕尖端的微压痕仪进行。该仪器的称重传感器由弹簧刚度为k = 49.94 N / m的双叶悬臂梁和检测范围为100 µm的电容式探头组成。根据测得的悬臂梁的垂直挠度和弹簧刚度,可以计算出压痕尖端的法向力。通过从双叶悬臂梁悬挂已知质量的砝码并使用电容探针测量产生的位移来执行生物压痕仪的校准。弹簧刚度是通过将一条线拟合到所得力与位移曲线上来确定的。为了与称重传感器保持一定的偏移量,并使其易于拆卸和清洁,压痕尖端安装在约15毫米长的尼龙螺纹杆上。压痕尖端与安装杆尖端粘合在一起,互补的安装螺母与称重传感器粘合在一起。为了控制压痕尖端的垂直位移,将称重传感器安装到PI压电平移z工作台上,该工作台的行程范围为250 µm,分辨率为1 nm。随后将平移z工作台安装到2轴线性平移工作台上,以使压痕尖端在XY平面中对齐。D3N公燕尾榫安装法兰固定在XY平移台的顶部,以使生物压痕仪能够安装在倒置显微镜的聚光镜槽中。生物压痕仪通过定制的Labview程序进行控制。该程序控制压电平台的垂直位移,同时记录来自电容传感器的信号,以计算双叶悬臂梁的垂直偏转。





图1.微压痕仪。A.使用安装在倒置显微镜上方的微压痕仪进行生物压痕测量。B.微型压痕仪包括一个半球形压痕头,一个用于测量法向力的测力传感器,一个具有纳米分辨率的垂直Z定位器,一个手动XY平移台以及一个燕尾榫安装法兰。C.压痕仪安装在倒置显微镜的聚光镜槽中,并用六角定位螺钉固定到位。



生物压痕仪安装在尼康Eclipse Ti2-E倒置显微镜上,该显微镜配有C2 +共聚焦成像系统和电动平移台。显微镜配置为使用488 nm激发激光和525/50二向色滤光片立方体捕获FITC荧光图像(图2A)。另外,在压痕测量之前,使用共聚焦反射显微镜将压痕尖端对准显微镜物镜正上方。对于反射成像,将488 nm激光用作光源,并且在检测器单元中安装了反射滤光镜立方体,以使所有光都可以到达检测器通道(图2B)。



图2 。显微镜光学配置。尼康Eclipse Ti2-E共聚焦显微镜配置为允许(A )在压痕测量过程中对腺泡结构进行荧光成像,或(B )在仪器设置过程中使用反射显微镜来确定压痕尖端的中心。



在压痕测量期间,使用定制的P etri盘支撑板固定 玻璃底部P etri盘,该支撑板安装在Nikon Eclipse Ti-2显微镜的板适配器中(图3)。玻璃底部P ETRI盘被定位在所述支撑板(1)的中心处的目标孔和使用两个安装臂(2)被牢固地固定。安装臂施加压力到P压痕测量期间ETRI盘盖以减轻运动。在P etri皿的盖子上钻一个孔,为生物压痕仪提供一个开口。为了在压痕测量过程中调节温度,该板上装有热电偶(3)和镍铬合金加热丝(4),它们连接到开关温度控制器上。设置电源的电源电压和电流设置,以维持所需的37°C温度,同时最大程度地减少开关周期的频率。

关键字:生物压痕, 微压痕, 生物力学, 组织机械力, 核力学, LINC 复合体, 腺泡发育



 

图3.培养皿支撑板。ģ小姑娘底部P ETRI菜肴使用定制支撑板压痕测量期间固定装配到显微镜的平移台上的板适配器。钍的电子温度P ETRI菜使用温度控制器,一个热电偶,以及一镍铬合金加热元件调节。
软件
 
以下软件用作此生物识别协议和数据分析过程的一部分:
尼康NIS-Elements AR成像软件(版本4.30.02)
National Instruments LabView(2014版)
FIJI ImageJ(https://imagej.net/Fiji)(版本1.52g)
Microsoft Excel(Office 365)
OriginPro绘图软件(8.5版)
 
程序
 
细胞培养和Acini生长
注意:Zhang等报道了细胞培养方案,细胞系发育和腺泡结构生长的完整描述。(2019)。
按照用于解冻,喂养和传代的标准细胞培养方案培养和维护MDCK II细胞。在潮湿的5%CO 2环境中于37 ° C孵育细胞。该协议的配方部分中提供了细胞培养基成分及其相应浓度。
在进行机械压痕测量之前一周,在8孔腔玻片中诱导MDCK II细胞的腺泡形成。
将Matrigel涂在8孔的Nunc Lab-Tek腔室玻片上,方法是在腔室玻片的每个孔中涂抹40-45 µl的Matrigel。在37 ° C下孵育至少30分钟,或直到凝胶完全固化。
按照标准细胞培养程序胰蛋白酶消化MDCK II细胞。将细胞以125 xg离心15分钟,然后除去上清液。将MDCK II细胞重悬于MDCK acini压痕培养基中。该方案的配方部分中提供了MDCK acini压痕培养基成分及其相应浓度。
用MDCK压痕培养基以〜5,000个细胞/孔的接种密度将MDCK II细胞接种到Matrigel包被的8孔室玻片中。在湿润的5%CO 2环境中于37 ° C孵育室载玻片约7天,或直到MDCK细胞已形成腺腺泡结构。
转移腺泡结构到玻璃底P ETRI菜为压痕测量。
涂覆玻璃底P ETRI盘用0.01重量%的聚-L-赖氨酸。地方200μl的0.01重量%的聚-L-赖氨酸到玻璃底P ETRI培养皿中并让坐在1个小时。除去聚-L-赖氨酸溶液,并用1x PBS溶液冲洗。聚-L-赖氨酸应防止在压痕过程中痤疮结构的任何打滑。
从基质胶生长基质中除去腺泡。在1 ° C PBS溶液中于4 ° C洗涤Matrigel包被的8孔腔玻片中的腺泡。在含有5 mM EDTA,1 mM NaVO 4的1x PBS溶液中孵育包含生长的腺泡的Matrigel包被的8腔室玻片。,以及1.5 mM NaF从腺泡结构中去除Matrigel生长基质。通过在4 ° C下以125 xg离心15分钟,从Matrigel支持基质中分离出腺泡结构。除去上清液,并用在4 ° C下制备的1x PBS溶液冲洗所得沉淀物。再次以125 xg的离心力离心结构15分钟,除去上清液。重悬沉淀成1×PBS中的溶液在4 ° C和腺泡转移到聚-L-赖氨酸包被的玻璃底P在〜1000腺泡/的接种密度ETRI菜肴P ETRI菜。它保持腺泡密度相对低的玻璃中的底部是很重要的P ETRI菜肴,以避免压痕实验期间接触多个腺泡结构。
用钙黄绿素AM染色acini结构,以检查单个acini的活力,并在压痕过程中对acini进行荧光共聚焦显微镜检查。用补充了1:2,000份钙黄绿素AM细胞染料的适当细胞生长培养基替换PBS溶液。在湿润的5%CO 2环境中于37 ° C孵育痤疮20分钟。除去培养基和钙黄绿素AM溶液,并替换为新鲜的细胞培养基。继续在潮湿的5%CO 2环境中于37 ° C孵育杆菌,直到进行压痕实验为止。
 
仪器设置
安装生物压痕仪和P ETRI盘支撑板到共焦显微镜。生物压痕仪通过冷凝器插槽安装在共焦仪上;将生物压痕仪的安装法兰滑入冷凝器插槽中,并拧紧安装螺钉,直到牢固地安装了生物压痕仪。将P etri皿支撑板放入共聚焦显微镜平移台上的板适配器中。将P etri盘支撑板连接至温度控制器和电源设备,并调整电源设置以保持37 ° C的温度。
在压痕尖端上涂上F-127普朗尼克,以减轻玻璃压痕尖端与acini结构之间的粘附作用。通过从安装螺母上松开安装螺钉,从双叶悬臂梁上卸下压痕尖端。用O 2等离子清洁器等离子清洁压痕尖端1分钟。等离子清洁后,立即将压痕尖端浸入0.1 wt %F-127普朗尼克溶液中10分钟。用1x PBS溶液冲洗压痕尖端,然后将压痕尖端重新安装到双叶悬臂梁上。
调整电容探头的高度,以确保双叶悬臂梁在电容探头的检测范围内。电容式探头应足够靠近以检测悬臂梁,但又要足够远,以使压痕期间悬臂梁的挠度保持在电容式探头的检测极限之内。
将压痕尖端对准显微镜物镜的中心。从最低幅度的物镜开始,降低压痕尖端并调整物镜焦点,直到压痕尖端处于焦点内。使用XY线性平移台,调整压痕尖端的位置,以使压痕尖端的中心与显微镜视场的中心对齐。将反射滤镜立方体安装到共聚焦显微镜的检测器单元中,然后将反射成像设置加载到NIS-Elements软件中(图2B)。调整共聚焦显微镜的焦平面以找到压痕尖端的顶点。继续将压痕尖端的中心与显微镜视场的中心对齐。
使用逐渐提高的物镜放大倍数重复对准步骤,直到压痕尖端居中位于20x显微镜物镜的正上方。建议在缩进期间使用1.5倍光学变焦功能。将压痕对准显微镜物镜后,从共聚焦显微镜的检测器单元上取下反射率立方体,重新安装438/24滤镜立方体,然后在NIS-Elements软件中选择DAPI / FITC / TRITC成像设置(图2A) )。建议此时检查压痕尖端是否有任何可能导致压痕尖端与acini结构之间不规则表面接触的碎屑。
固定玻璃底P上的共焦显微镜的平移台上ETRI菜。放置玻璃底P含有腺泡结构到ETRI盘P ETRI盘支撑板。使用两个安装臂将P etri盘固定到支撑板上。
确定P etri皿的玻璃表面的位置,并将压痕仪的力和位移值清零。导航到P etri盘中央的区域,显微镜视野中没有痤疮。启动压痕软件并将力和位移测量值清零。使用压电z平台将压痕尖端朝着P etri碟的表面驱动100 µm ;此时,压痕尖端不应与表面接触,并且力的测量值应在0 N左右波动。使用聚光镜台的焦距调节手动降低压痕仪,直到压痕尖端与玻璃表面接触可以从测得的力突然而急剧地增加,以及力测量中的噪声降低中看出。一旦与表面接触,请使用聚光镜台的焦距调节缓慢升高压痕仪,直到压痕尖端不再与表面接触,并且测得的力返回到0N。将压痕尖端提高100 µm使用压电z平台。现在,压痕尖端应位于P etri皿的玻璃表面上方约100 µm的位置–足够高,可避免在移动共聚焦显微镜的平移台时与痤疮结构接触,但应足够低以在压痕测量期间实现接触。
 
Acini结构的机械压痕
进行腺腺体结构的机械压痕,以确定腺体结构发生机械性破坏的平均施加压力。压痕尖端以固定的压痕速率缓慢下降至与单个腺泡结构接触,同时通过细胞的同时荧光成像记录所施加的力。当压入力超过测得力中的背景噪声水平时,观察到的腺泡结构变平坦。进一步增加在腺泡持续变形所施加的力的结果,直到有导致细胞泡和/或细胞的破裂(图的形成的结构坍塌URE 4A)。这些破裂事件在变形腺泡使我们能够查明在该第一破裂事件发生的时间(图11的荧光图像捕获URE 4B)。初始断裂后,压痕尖端会继续推入腺结结构,直到测得的施加载荷达到指定的最大压痕力为止。一旦达到最大压入力,压入尖端将以指定的压入速率拉出结构,返回到起始z位置。要执行acini结构的机械压痕:
找到一个隔离的痤疮结构,并将其在显微镜视场的中心对准压痕尖端的正下方。重要的是,在显微镜的视场内隔离痤疮结构,以避免与其他痤疮结构接触。建议在放置腺泡结构时使用明场成像,以避免腺泡结构长时间暴露于共聚焦激光和荧光染料的光漂白。
Acini结构的未对准可能导致压痕尖端与acini之间的表面接触不规则,导致压痕尖端在acini结构破裂之前与玻璃基板接触,从而导致力测量不正确。力与位移曲线的突然近乎垂直的增加是一个强烈的信号,表明acini结构没有正确地与压痕尖端对齐。
将共焦显微镜的焦平面导航到所需的成像平面以进行压痕测量。它建议编选择一个焦平面接近于腺泡结构的底部完全捕捉期间压痕过程(图4A)的变形; 如果焦平面选择得太高,可能会导致破裂结构在发生破裂事件之前从成像平面中变形出来。
注意:这是可选的,此时执行共焦z扫描以在压痕测量之前捕获acini结构的形状。
准备共焦显微镜用于压痕测量。在共聚焦软件中启动完美聚焦,并调整共聚焦软件的目标区域和捕获延迟,以实现最快的成帧速度。在压痕测量过程中,较高的帧速率将在确定Acini结构的破裂压力时改善时间分辨率。
准备用于压痕测量的压痕仪。在压痕仪软件中,设置压痕率和最大压痕力的参数。我们建议压入速度为0.1 µm / s,最大压入力为100 µN。更快的压痕速率将降低整个测量过程中捕获的荧光图像的时间分辨率。当确定破裂压力时,较慢的压入速度会改善时间分辨率,但会增加实验时间和细胞的激光照射时间。最大压入力应设置得足够高,以使acini结构在达到最大力之前就破裂。根据我们的经验,最大压痕载荷为100 µN,足够高,足以引起痤疮破裂。但是,更高的最大压痕力不会改变测量结果。
同时在共聚焦显微镜上开始对痤疮结构的压痕测量和成像延时。记录压痕测量开始时间和成像延时中的所有差异。
 
数据分析
 
一旦压痕完成,从压痕仪测量的法向接触力和荧光图像测得的接触面积就确定了腺泡结构的破裂压力。
 
使用共聚焦显微镜(图4B)捕获的延时图像确定痤疮结构首次破裂的时间。校正压痕测量的开始时间和共焦延时之间的任何时间差。
在首次观察到破裂事件的时刻测量acini结构的接触面积(图4B)。可以使用NIS Elements软件或FIJI ImageJ来完成。接触面积可以通过在破裂时在acini结构周围绘制最佳拟合圆来测量。
通过破裂时的痤疮结构确定正常的接触力体验。可以使用Microsoft Excel或OriginLabs绘图软件来完成此步骤。将力对时间数据加载到绘图软件中,并在先前确定的断裂时间记录压入力。
根据所测得的接触面积和所测得的法向接触力计算acini结构的破裂压力。球的赫兹接触的平均接触压力为< P > = F / A ,其中F是从压痕仪测得的法向力,A是从共焦图像测得的接触面积。
 

图4.来自微压痕测量的数据分析。A.生物压痕实验的示意图。选择成像平面靠近Acini结构的底部,以捕获压痕测量过程中的全部变形。B.通过共聚焦显微镜捕获的荧光图像显示整个压痕中的腺泡结构的变形。随着力的增加,可以在随后的帧之间观察到细胞泡的形成和/或细胞的破裂,这表明了腺泡结构的破裂。在给出的示例中,我们观察到t = 812 s时痤疮破裂,这可以通过细胞破裂和钙黄绿素染料释放到周围介质中来看出。在观察到破裂的情况下,通过手动将圆形拟合到痤疮结构来确定接触面积。Ç 。断裂时的压入力由生物压入仪器的力测量值确定。(µN:微牛顿)
 
讨论区
生物压痕为探索生物材料的材料特性以及细胞系统对外部作用力的集体行为提供了一种手段。通过将机械压痕与荧光图像耦合,我们直接观察到由外部施加的机械力导致的单个腺腺泡结构的变形和破裂。开发该协议是我们探索LINC复合体在腺腺泡结构的发展和维持中所起的作用的一部分(Zhang等,2019)。由具有破坏的LINC复合物的MDCK II细胞形成的Acini在比施加由完整LINC复合物形成的那些更高的施加压力下失效。这些结果似乎与观察到的具有破坏的LINC复合物的腺泡结构中充满的管腔一致。此外,用Y-27632处理LINC破裂的痤疮可将破裂压力恢复到未破裂的水平。
  尽管此处介绍的协议侧重于对单个棘突结构进行生物压痕测量,但是可以开发类似的压痕协议来探索各种生物系统。例如,已经使用类似的微压痕方案研究了MDCK单层的弹性模量和渗透性,以及破坏3T3成纤维细胞核包膜所需的最低压力(Schulze等人,2017; Halfmann等人,2019)。此外,可以通过添加第二个称重传感器来测量横向力来研究剪应力和摩擦对生物系统的影响(Urueña等人,2018)。继续探索生物系统对外力和机械排队的反应对理解集体细胞行为和发育生物学至关重要。
 
菜谱
 
以下是用于培养MDCK细胞和执行生物识别方案的各种解决方案的配方:
MDCK细胞生长培养基
DMEM培养基
4.5 g / L葡萄糖
10%v / v DBS
MDCK acini压痕介质
DMEM培养基
4.5 g / L葡萄糖
10%v / v供体牛血清
2%v / v Matrigel基质基底膜
基质胶洗涤液
1x PBS溶液
5毫米EDTA
1毫米Na 3 VO 4
1.5 mM NaF
阿奇尼染料培养基
细胞生长培养基
钙黄绿素(1:2000)
F-127普朗尼克解决方案

0.1 wt %F-127普朗尼克
 
致谢
 
这里介绍的缩进协议是Zhang等人研究工作的一部分。(2019)。这项工作得到了TP Lele的NIH(授予R01 EB014869)的支持和TE Angelini的NSF (授予DMR-1352043)的支持。作者要感谢Kyle D. Schulze博士和W. Gregory Sawyer博士在开发此协议中使用的微压痕仪方面所做的贡献。作者还要感谢Jennifer Lippencott-Schwart博士慷慨捐赠了MDCK II细胞。
 
利益争夺
 
作者没有任何经济利益冲突可宣布。
 
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引用:O'Bryan, C. S., Zhang, Q., Lele, T. and Angelini, T. E. (2020). Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument. Bio-protocol 10(23): e3847. DOI: 10.21769/BioProtoc.3847.
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