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Mar 2019
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Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices
小鼠离体肺组织切片中肺泡生成的延时成像   

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Abstract

Alveoli are the gas-exchange units of lung. The process of alveolar development, alveologenesis, is regulated by a complex network of signaling pathways that act on various cell types including alveolar type I and II epithelial cells, fibroblasts and the vascular endothelium. Dysregulated alveologenesis results in bronchopulmonary dysplasia in neonates and in adults, disrupted alveolar regeneration is associated with chronic lung diseases including COPD and pulmonary fibrosis. Therefore, visualizing alveologenesis is critical to understand lung homeostasis and for the development of effective therapies for incurable lung diseases. We have developed a technique to visualize alveologenesis in real-time using a combination of widefield microscopy and image deconvolution of precision-cut lung slices. Here, we describe this live imaging technique in step-by-step detail. This time-lapse imaging technique can be used to capture the dynamics of individual cells within tissue slices over a long time period (up to 16 h), with minimal loss of fluorescence or cell toxicity.

Keywords: Imaging alveologenesis (肺泡生成成像), Time-lapse imaging (延时成像), Deconvolution software (反卷积软件), Precision-cut lung slices (离体肺组织切片), Lung development (肺组织发育)

Background

Prenatal and postnatal lung development is classified into several distinct stages beginning with budding from the foregut endoderm followed by branching morphogenesis, sacculation and alveologenesis within surrounding lung mesenchyme. This developmental process is tightly regulated by a well-orchestrated signaling programme and cellular components (Kotton and Morrisey, 2014; Akram et al., 2016). The major function of the lungs is gas exchange, which occurs via diffusion (Herriges and Morrisey, 2014). This diffusion takes place between the thin cellular layers of alveolar epithelium and capillary endothelium (Roth-Kleiner and Post, 2005). Dysregulated alveologenesis is linked with a number of neonatal and infant diseases, including bronchopulmonary dysplasia (BPD) and pulmonary hypoplasia (Kreiger et al., 2006; Hilgendorff et al., 2014). In adults, alveolar damage is a component of several chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Currently, there are no curative treatments for these diseases other than lung transplantation (Warburton et al., 2006; Madurga et al., 2013; McGowan, 2014) and there is an unmet need to understand the mechanisms of alveologenesis in order to develop effective treatments.

In mice, sacculation begins around embryonic day 17.5 and is followed by alveologenesis, which begins within the first few days of postnatal life and is mostly completed in first month of life (Herriges and Morrisey, 2014). However, the most active phase of alveologenesis occurs in first two weeks of postnatal life with the majority of alveoli formed by postnatal day (P) 21 (Hind et al., 2002; Snoeck, 2015).

Current understanding, based on static imaging experiments, is that alveologenesis occurs through repeated septation events that sub-divide primary air sacs to increase the number and surface area of alveoli (Amy et al., 1977; Mund et al., 2008). Real-time visualization of alveologenesis is challenging due to their location deep inside the body and the relatively slow duration of this process. A recent study used both ex vivo and in vivo live-imaging to study the sacculation stage of mouse lung development, immediately prior to alveologenesis, but these techniques are not suitable for imaging postnatal lungs (Poobalasingam et al., 2017; Li et al., 2018).

Precision cut lung slices (PCLS) contain intact alveoli and are increasingly used to study lung biology and disease pathogenesis (Meng et al., 2008; Sanderson, 2011; Thornton et al., 2012). Time-lapse imaging of PCLS has been used to show dynamic interactions of mesenchymal cells and macrophages with the extracellular matrix in adult normal and fibrotic mouse lungs, as well as in PCLS of human lungs (Burgstaller et al., 2015). In addition, quantifiable ex vivo alveologenesis has been demonstrated in early postnatal mouse PCLS culture (Pieretti et al., 2014). Using a combination of widefield microscopy and image deconvolution on postnatal mouse PCLS we have developed a method to capture the morphological mechanisms of alveologenesis in real-time (Akram et al., 2019). Here we describe the detailed protocol for real-time live imaging of postnatal alveologenesis.

Materials and Reagents

  1. 50 ml centrifuge tubes (Thermo Fisher, catalog number: 338652)
  2. Metallic spatula (Fisher Scientific, catalog number: 11523482)
  3. Glass coverslips (Thermo Fisher, catalog number: 102260)
  4. Probe Point (Blunt) needles, 25 G, 19 mm (0.75 inch) (Harvard Apparatus, catalog number: 725461) for P3 mice; Monoject blunt needles with Aluminum Hub, 23 G, 1 inch (Harvard Apparatus, catalog number: 722349) for P7 mice, and 21 G (Custom made from 21 G syringe needle) for P14 and adult mice (all from Harvard Apparatus UK)
  5. 24-well plate (Corning® Costar® TC-Treated Multiple Well Plates) (Sigma-Aldrich, CLS3527-100EA)
  6. 96-well plate (Thermo Fisher, catalog number: 249952)
  7. Ibidi 24-well μ-plate (Uncoated) (Ibidi, catalog number: 82401)
  8. Transwell (0.4 µm pore, 12 mm, polyester membrane) (Corning, catalog number: 3460)
  9. Microscope slides, SuperFrost®, Menzel Gläser (VWR, catalog number: 631-1318)
  10. Non-sterile silk black braided suture spool, 22.9 m, Size 5-0 (Harvard Apparatus UK, catalog number: 517607)
  11. BD Micro-Fine+ 29 G, 1 ml Insulin Syringes (MediSuuplies, catalog number: PMC3743)
  12. Syringes, 5 ml (VWR International, catalog number: SART16644-E)
  13. Thermo ScientificTM NuncTM Cell Culture/Petri Dishes, 100 mm (Fisher Scientific, catalog number: 10508921)
  14. Swann-Morton Surgical scalpels, No.22 (MediSupplies, catalog number: PMC0105)
  15. Wet ice and ice box
  16. C57BL/6 male and female mice from Charles River Laboratories
  17. EpCAM-FITC (CD326) monoclonal antibody (eBioscience, catalog number: 11-5791-80; Clone G8.8)
  18. Alexa-647 conjugated PECAM antibody (CD31-Alexa 647) (Biolegend, catalog number: 102416; Clone 390)
  19. Laboratory tissue (Blue roll)
  20. Metal flat washer (weight 1.66 g) (M8-5/16th inches diameter) (B&Q, UK)
  21. Absolute ethanol (Sigma-Aldrich, catalog number: 34852-M)
  22. Pentobarbitone (Pentoject, Animalcare, catalog number: XVD 132)
  23. Low-melting-point agarose (Sigma-Aldrich, catalog number: A9414)
  24. Hanks' Balanced Salt Solution (HBSS) (1x) (Life Technologies, catalog number: 14025-050)
  25. HEPES 1 M (Life Technologies, Gibco, catalog number: 15630080)
  26. Phosphate Buffered Saline (PBS) (Life Technologies, Gibco, catalog number: 20012068) 
  27. Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Gibco, catalog number: 31966-021), also referred to as 'DMEM basal media” in the Recipes
  28. Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Gibco, catalog number: 15140122)
  29. MTT reagent (Thiazolyl Blue Tetrazolium Bromide) (Sigma-Aldrich, catalog number: M2128)
  30. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855)
  31. 10% neutral buffered formalin (Sigma-Aldrich, catalog number: HT501128)
  32. DAPI (Stock concentration 10 mg/ml) (Sigma-Aldrich, catalog number: D9542)
  33. LIVE/DEAD® Viability/Cytotoxicity Kit (Thermo Fisher Scientific, catalog number: L3224)
  34. Methanol (Sigma-Aldrich, catalog number: 34860)
  35. ProLong® Gold Antifade Mountant (Thermo Fisher Scientific, catalog number: P36930)
  36. Silicon rhodamine far-red fluorophore-conjugated DNA minor groove binder bisbenzimide (SiR-DNA) (tebu-bio Ltd, catalog number: SC007)
  37. Phenol red-free DMEM with HEPES (Life Technologies, catalog number: 21063029), also referred to as 'Phenol red-free DMEM with HEPES basal media” in the Recipes
  38. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7030)
  39. Triton X-100 (Sigma-Aldrich, catalog number: X100)
  40. (Optional) para-Nitroblebbistatin (Cayman Chemical Company, catalog number: 13891) 
  41. (Optional) Cytochalasin D (Sigma-Aldrich, catalog number: C8273)
  42. HBSS/HEPES ice cold buffer (see Recipes)
  43. Agarose solution (see Recipes)
  44. SF-DMEM (see Recipes)
  45. Image media (see Recipes)
  46. MTT working solution (see Recipes)
  47. 70% ethanol (see Recipes)
  48. 70% methanol (see Recipes)

Equipment

  1. Curved dissecting forceps, 10 cm, Serr/C (World Precision Instruments Ltd, catalog number: 15915)
  2. Fine tip dissecting scissors, 10 cm, straight (World Precision Instruments Ltd, catalog number: 14393)
  3. Surgical scissors, 14 cm, straight (World Precision Instruments Ltd, catalog number: 14192)
  4. Spring scissors, 12 cm straight, 12 mm extra-fine blades (World Precision Instruments Ltd, catalog number: 14125)
  5. Stainless steel blades for vibratome (Campden Instruments LTD, catalog number: 7550-1-SS)
  6. Automated vibratome (Compresstome® VF-300-0Z; Precisionary Instruments LLC, USA)
  7. Incubator (Humidified, 37 °C, 5% CO2)
  8. -20 °C freezer
  9. GFP filter, excitation 450-490 nm, emission 500-550 nm (for EpCAM-FITC)
  10. Cy-5 filter, excitation 625-655 nm, emission 665-715 nm (for SiR-DNA and PECAM)
  11. Plate reader (Tecan; SunriseTM, INSTSUN-1)
  12. Zeiss Axio Observer inverted widefield microscope, with Lumencor Spectra X LED light source and Hamamatsu Flash 4.0 camera (Zeiss, Germany)
  13. Zeiss LSM-510 inverted confocal microscope (Zeiss, Germany, model: LSM 510)
  14. Leica DM2500 widefield microscope (Leica Microsystems, model: Leica DM2500)

Software

  1. Zen2 acquisition software, blue version (Zeiss, Germany)
  2. ZEN 2009 (black edition) software (Zeiss, Germany)
  3. FIJI (ImageJ, version 2.0)
  4. Icy open source bioimaging analysis software (Version 1.9.8.0; created by the Quantitative Image Analysis Unit at Institut Pasteur, Paris, France)
  5. Huygens deconvolution software (Scientific Volume Imaging, SVI, Essential version 17.10)
  6. NIS-Elements (Version 4.50, Nikon Instruments, UK)
  7. GraphPad Prism version 5
  8. Microsoft Excel (Microsoft Office 2011 version)
  9. Microsoft PowerPoint (Microsoft Office 2016 version)

Procedure

  1. Producing precision-cut lung slices
    1. Mouse dissection and lung harvesting
      1. Humanely kill postnatal day 3 (P3), P7, P14 or adult (6-8 weeks) C57BL/6 mice by intraperitoneal injection of pentobarbital (50 mg/kg body weight). Inject 25 µl of pentobarbital for P3, 50 µl for P7, 75 µl for P14 and 100 µl for adult mice using 29 G, 1 ml Insulin Syringes (Figure 1A7). Mice will die in 5-10 min after injection. Confirm death by exsanguination of femoral artery.
      2. Place the mouse on a dissection tray in supine position. Spray 70% ethanol over the anterior aspect of the abdomen, chest and neck.
      3. Using curved forceps (Figure 1A2) to pinch the skin from the anterior abdominal wall and gently pull it up, cut a small piece of skin leaving the inner peritoneum intact.
      4. Using a pair of fine scissors (Figure 1A4) make a longitudinal incision along the midline of anterior abdomen and chest wall and extend it up to the chin (Figure 1B).
      5. Using two pairs of curved forceps, retract the skin on both sides to expose the anterior aspects of the abdomen, chest wall and neck. (Figure 1Ci) (Video 1).
      6. Using curved forceps carefully lift the peritoneal wall at the upper part of the abdomen, just below the costal margin and make a small opening using the tip of the fine scissors.
      7. Extend the opening to the right and left using fine scissors. This will expose the diaphragm.
      8. Gently lift the lower end of the sternum and make an opening on the upper part of diaphragm using the tip of the fine scissors. Then extend the opening to both side of the diaphragm along the inner surface of the costal margins (Figure 1C i, ii, yellow dotted line). At this stage you will be able to see pale pink colored right and left collapsed lungs (Figure 1C iii, arrow).
      9. Lift the chest wall by holding the lower end of the sternum with curved forceps and carefully make a parasternal incision (to the left and right) to separate anterior chest wall from the body (Figure 1C i, iii, yellow dotted line) (Video 1).
      10. Using curved forceps and fine scissors, remove muscle and fascia from the anterior and lateral aspects of the neck wall.
      11. Using curved forceps, make an opening between the posterior aspect of the trachea and the anterior wall of the esophagus at the midpoint of the neck (Video 1).
      12. Insert a piece of sterile suture through the opening and place it perpendicular to the trachea at its midpoint (Figure 1).
      13. Make a small opening in the anterior wall of the trachea just below the cricoid cartilage using fine tipped straight spring scissors (Figure 1A, (6), 1C (iii) yellow arrow, Figure 1D (ii) inset).
      14. Carefully insert a rigid metallic cannula 25 G for P3, 23 G for P7 and 21 G for P14 and adult mice through the trachea up to a millimeter above the bifurcation of the principal bronchi and fix in place with a suture (Figure 1C (iv) and 1D (ii) inset). The suture was tied, with moderate tightness, around the trachea including cannula inside, by applying two knots.
      15. After cannulation, inflate the lungs by gently injecting 1.5% low-melting-point agarose prepared with HBSS/HEPES buffer warmed to 37 °C (see Recipes). Use a 5 ml syringe to instill the agarose and keep the syringe attached to the cannula until the agarose solidifies. Inflate both lungs keeping them in situ within the chest cavity using 0.2 ml for P3, 0.275 ml for P7, 0.35 ml for P14 and 1 ml for adult lungs. These volumes enable the lungs to be fully inflated without hyper- or sub-optimal inflation (Figures 1C and 1D).
      16. After inflation, place a piece of double layered laboratory blue roll over the exposed chest wall and put some wet ice on to the blue roll to solidify the agarose.
        Note: Depending on mouse age, this will take 1 to 2 min.
      17. Remove the cannula by gently pulling it out from the trachea and tighten the suture knot.
      18. Excise the trachea from its upper (mouth) end. Lift the trachea by holding the free ends of the suture and gently remove the entire heart and lungs from the chest cavity (Figures 1C and 1D).
      19. Immerse the whole heart and lungs in ice-cold serum-free DMEM (SF-DMEM) in a 50 ml centrifuge tube, secure the cap and store on wet ice until slicing. The tissue can be kept on ice for 2-3 h.

        Video 1. Mouse dissection: from skin incision to trachea mobilization. (All animal maintenance and procedures were carried out according to the requirements of the Animal (Scientific Procedures) Act 1986. Animal work was approved by the South Kensington and St. Mary’s AWERB committee, Imperial College London.)


        Figure 1. Mouse dissection and lung harvesting workflow. A. Dissection equipment: 1. Suture, 2. Curved forceps, 3. Fine tip forceps, 4. Fine tip dissecting scissors, 5. Surgical scissors, 6. Spring scissors, 7. Pentobarbital in 1 ml syringe attached with hypodermic needle. B. Skin incision line. C. Adult mouse dissection, agarose inflation and harvesting of lungs. D. Postnatal day 3 (P3) mouse dissection, agarose inflation and harvesting of lungs.

    2. Precision-cut lung slicing
      1. Prepare the automated vibratome: Sterilize the buffer tray of the vibratome thoroughly by spraying with 70% ethanol and leave to dry (Figure 2A). Attach vibratome blade to the blade holder using super glue and leave to dry. Sterilize specimen holder and specimen tube using 70% ethanol spray and air dry. Place the syringe chilling block in -20 °C freezer for 15-30 min to cool it down prior to use (Figure 2B).
      2. Have ready approximately 30 ml of 1.5% agarose gel made with HBSS/HEPES at 37 °C temp. Prepare 200 ml of sterile ice cold 1x HBSS/HEPES buffer and keep on ice. Prepare a 24-well plate with 1 ml ice cold, sterile SF-DMEM per well and keep on ice.
      3. In a 10 cm diameter Petri dish, separate the left lung lobe from the left bronchus using a scalpel blade (Figures 2C and 2D).
      4. Excise a tiny section of tissue from the basal end of the lung using the scalpel blade (Figure 2D; outline of the tissue excised is marked with dotted line).
        Note: This helps the lobe to sit on the specimen holder vertically.
      5. Place the lung lobe on a piece of clean laboratory blue roll to soak up excess media (Figure 2D).
      6. Place a tiny drop of super glue onto the cutting end of the specimen holder (Figure 2Ei) and gently place the cut end of the lung lobe onto the glue drop, hold it vertically with curved forceps until the lung lobe tightly adheres to the specimen holder (Figure 2Eii).
        Note: It takes 15-30 s to attach the lung lobe onto the specimen holder.
      7. Insert the specimen holder into the metal specimen tube, hold it vertically keeping the lung lobe inside the tube and facing vertically up (Figure 2Eiii).
      8. Using a 5 ml syringe, fill the specimen tube with 37 °C warm 1.5% agarose so that the entire lobe is submerged in agarose (Figure 2Eiii).
      9. Place the cold syringe chilling block (Figure 2B) around the specimen tube and hold it in place until the agarose tissue block solidifies (Figure 2Eiv).
        Note: It takes about a minute to solidify the agarose.
      10. Insert the specimen tube into the buffer tray and align blade and cutting end of the agarose tissue block.
      11. Fill the buffer tray with ice cold HBSS/HEPES buffer. Set the desired thickness of tissue slices to 300 µm. Set the cutting speed to number 5 and cutting oscillation frequency to 5. Operate the vibratome in ‘continuous mode’.
      12. Start collecting transverse precision-cut lung slices (PCLS). Remove the lung slices from the first 1/3rd of the lung lobe from the buffer tray using a small metallic spatula. Collect the slices from the middle 1/3rd of the lobe and place them in the wells of a 24-well plate containing ice cold SF-DMEM (Figure 2F). The middle third of the lung lobe provides almost equal sized slices. Take 1 lung slice in each well in 1 ml media. (Using this technique, a P3 left lung provides 12 slices and an adult lung provides approximately 36 slices.)
        Note: It is important to use equal sized slices for imaging and metabolic assays, the remaining slices can be used for additional assays, e.g., immunostaining, protein or RNA extraction.
      13. Incubate PCLS at 37 °C in the presence of 5% CO2 for 2 h and then wash twice with warm SF-DMEM to remove excess agarose from around the tissue in sterile conditions, under the flow hood.
        Note: Some agarose will remain in the airspaces within the lungs and this is important to retain lung architecture during culture.
      14. Incubate the PCLS for a further 1 h in SF-DMEM at 37 °C in the presence of 5% CO2. From this point the PCLS are ready for live imaging and further experimentation.
        Note: Use the samples immediately for imaging (and migration analysis); but slices can be kept in culture overnight for other experimentation.

  2. Live cell imaging of lung alveologenesis
    1. Label-free, bright-field live imaging
      To visualize gross structural changes in lung slices over time, live bright-filed imaging can be performed as follows:
      1. Use phenol red-free DMEM and ibidi 24-well μ-plate for live imaging.
      2. Prepare transwells for imaging: using scissors cut and remove the rim of the transwell so that the transwell filter comes into contact with the lung slice at the bottom of the well (Figure 2G). Wash the transwells with HBSS (warm) to remove debris and place them in unused wells of ibidi 24-well μ-plate without buffer.
      3. Add 50 µl of warm (37 °C) phenol red-free SF-DMEM to the center of a well of the ibidi μ-plate.
      4. Using a thin sterile metallic spatula take a PCLS from Step A2n above and carefully place at the center of the well of ibidi μ-plate. Make sure that the lung slice is placed in the center and is flat and not folded.
      5. Then gently place the modified transwell on top of the PCLS.
      6. Add 500 µl phenol red-free SF-DMEM to the upper chamber and 300 µl to the bottom chamber of the transwell.
      7. To keep the PCLS in place, put a 1.66 g metal flat washer with a central hole on top of the transwell housing (Figures 2H and 2I).
        Note: This weight puts light pressure on tissue slice through the permeable transwell membrane, which holds the slice in place without damaging the tissue. The metal flat washer weight should not exceed 3.32 g.
      8. Incubate the PCLS at 37 °C in presence of 5% CO2 and 95% air (~21% O2) in a humidified incubator for 2 h. This incubation step allows the lung slice to settle down prior to image acquisition.
        Note: Similar sized PCLS should be allocated for MTT viability assay after live images, as shown in Figure 3.
      9. At the same time, humidify and pre-equilibrate the incubator chamber of an inverted Zeiss Axio Observer widefield epifluorescence microscope (or similar) for 30 min to 1 h with the following conditions: 37 °C, 5% CO2 and room air oxygen levels, approx. 21%.
      10. After incubation and pre-equilibration transfer the ibidi 24-well plate containing PCLS to the microscope incubator and maintain the incubator chamber conditions as above throughout the entire time-lapse duration.
      11. Start capturing time-lapse images using a long working distance 40x (0.7 NA, air) objective lens under bright-light (Figure 2I).
      12. For bright-field imaging, find the best focused plane at the region of interest and keeping this point as the center select 30 z-slices towards the upper surface of the slice and 30 slices towards the bottom surface of the slice with 1 µm step-gap between the slices (a total of 60 slices in the middle 60 µm z-axis of tissue).
      13. Set the time-lapse image capture interval to every 15 min for 12-19 h.
        Note: Four images per hour (15 min interval) reduces light exposure, hence minimizes fluorophore bleaching but produces smooth time-lapse video with 10-12fps (see, Supplementary Movie 2 in Akram et al, 2019).
      14. Select 4 separate fields of interest within alveolar regions, avoiding airways, from the four quadrants of a PCLS.
        Note: The 2 h incubation and pre-equilibration of the microscope incubator are crucially important to avoid the plane of focus drifting during time-lapse image acquisition. Image 4 separate PCLS per experiment.


        Figure 2. Precision-cut lung slicing and live imaging setup. A. Lung slicer: Vibratome, Compresstome® VF-300-0Z. B. Syringe chilling block for solidifying agarose. C. Agarose inflated lungs harvested from a P3 mouse. D. Separated left lung lobe on laboratory tissue roll. E. Schematic diagram showing agarose embedding of left lung lobe on specimen holder for making PCLS. F. Precision-cut lung slices are in 24-well plate. G. Modification of transwell for stabilizing lung slices on well plate. H. Precision-cut lung slices are set in ibidi 24-well μ-plate using modified transwells and metal flat washer (black rim). I. Schematic showing the relative position and alignment of metal flat washer, transwell, lung slice, and 40x long working distance air objective lens on an inverted microscope for live imaging.

    2. Live fluorescence imaging of immunostained PCLS
      Dual staining of PCLS for live imaging
      1. To visualize and track epithelial cells within the PCLS, dual stain cells with FITC-conjugated EpCAM antibody (EpCAM-FITC) and SiR-DNA. (EpCAM selectively labels epithelial cell membranes. SiR-DNA is silicon rhodamine far-red fluorophore-conjugated DNA minor groove binder bisbenzimide and stains the nuclei of cells).
      2. Incubate PCLS (from Step A2n) for 1 h at 37 °C with EpCAM-FITC antibody diluted 1:200 and SiR-DNA diluted 1:300 in DMEM (without shaking). Use 500 µl of antibody/SF-DMEM media for each well per PCLS.
      3. To visualize epithelial and endothelial cells within the alveolar region, incubate PCLS for 1 h at 37 °C with dual Alexa-647 conjugated PECAM antibody and EpCAM-FITC antibody both at 1:200 in 500 µl SF-DMEM per sample per well.
      4. After incubation, wash the PCLS three times with warm SF-DMEM.

      Time-lapse fluorescence imaging of PCLS
      1. Image media: Prepare image media by adding EpCAM-FITC antibody at 1:500 and SiR-DNA at 1:1000 for epithelial cells or PECAM at 1:500 along with EpCAM-FITC (1:500) for dual-labeling of epithelial/endothelial cells in 800 µl phenol red-free SF-DMEM for each PCLS per well.
        Note: SiR-DNA at 1:1000 dilution can be used as a nuclear marker for cells that are dual labeled with epithelial or endothelial markers.
      2. Place a 50 µl drop of image media at the center of a well in an ibidi 24-well μ-plate and carefully place a dual stained PCLS onto the image media using a thin metallic spatula.
      3. Place a modified transwell on top of the PCLS, add 500 µl of image media to the upper chamber and 300 µl image media to the bottom chamber and finally stabilize the transwell by placing a flat metal washer onto the transwell as described above (Figures 2H and 2I).
      4. Incubate the PCLS at 37 °C in presence of 5% CO2 and 95% air in a humidified incubator for 2 h. This incubation step intensifies the staining and allows the lung slice to settle down prior to image acquisition.
      5. Pre-equilibrate the microscope incubator chamber as described for bright-field imaging (as Step B1i).
      6. After incubation and pre-equilibration transfer the 24-well plate to an inverted Zeiss Axio Observer widefield epifluorescence microscope incubator and maintain the incubator conditions as above throughout the time-lapse duration.
      7. Start recording time-lapse images using a long working distance 40x (0.7 NA, air) objective lens using a GFP filter, excitation 450-490 nm, emission 500-550 nm (for EpCAM-FITC) and Cy-5, excitation 625-655 nm, emission 665-715 nm (for SiR-DNA and PECAM) from 4 fields of alveolar regions per slice for 8-19 h at 15 min intervals. Capture 11 images from the middle 11 µm thickness of tissue along the z-axis with a 1 µm step-gap between each slice to make a z-stack from each PCLS.
        Notes:
        1. Perform time-lapse imaging on four PCLS at a time per experiment. Do not capture bright-field images and fluorescent images from the same PCLS within an experiment. Exposure of bright light significantly bleaches the intensity of fluorescent staining. Do not use a UV or DAPI filter (excitation 358 nm and emission 463 nm) for live imaging of PCLS as this causes significant cell death. Zen2 acquisition software, blue version was used for both brightfield and fluorescence live imaging. 
        2. During live cell imaging, cellular dynamics or other behaviors can be manipulated by adding various factors such as cytokines or small molecules inhibitors into the image media. The resulting cell responses can be recorded in real-time. For example, we recently added the actin cytoskeleton modifiers Blebbistatin and Cytochalasin-D to PCLS and assessed their effects on cell migration using this live-PCLS imaging technique (Akram et al., 2019).
        3. To ensure the viability of PCLS, perform a viability assay on PCLS after every time-lapse live imaging experiment. For bright-field imaging on unlabeled PCLS perform Live/Dead staining and for fluorescence imaging on dual stained PCLS, perform an MTT assay.
        4. Duration of experiment p: From mouse dissection to the start of time-lapse imaging takes 7-8 h.

  3. Cell viability assays on PCLS after live cell imaging experiments
    1. MTT assay
      1. Place four PCLS that have undergone live imaging into an ibidi 24-well μ-plate, 1 slice per well. Place four similar sized, non-imaged PCLS that have been kept in an incubator with 37 °C, 5% CO2 for the same duration as the imaged slices in a conventional 24-well plate, 1 slice per well in phenol red-free SF-DMEM. Four similar-sized slices were selected to perform MTT assay to determine cell viability at the start of time-lapse imaging experiments (Figure 3).
      2. To assess metabolic activity within cells at the initiation of live imaging (0 h control), add 500 µl of 10% MTT solution (Stock Con. 5 mg/ml) made with SF-DMEM to each well.
        Note: This MTT needs to be done before starting live imaging (Figure 3B).
      3. Incubate the PCLS at 37 °C in presence of 5% CO2 for 1 h (Figure 3C).
      4. Remove the MTT media from the wells.
      5. Solubilize the formazan crystals formed within the viable cells by adding an equal volume of DMSO (500 µl) and incubating at 37 °C for 10 min.
      6. Take 200 µl of eluted formazan solution from each PCLS and place into individual wells of a 96-well plate (Figure 3D).
      7. Measure the absorbance (OD) at 570 nm and correct at 690 nm using a plate reader.
      8. To assess metabolic activity within post-time lapse PCLS, carefully remove the transwells after completion of time-lapse imaging. If PCLS adhere to the under surface of the transwell PET membrane, flush with warm HBSS using a pipette, to detach it. 
      9. Wash the PCLS with warm HBSS once.
      10. Then perform MTT assay on PCLS undergone time-lapse imaging and non-imaged PCLS cultured in a conventional incubator in the same way as described for initial time-point control PCLS above.
      11. Compare the initial time-point (0 h) and post-time lapse (Imaged and non-imaged PCLS) OD values to evaluate cell viability after time-lapse imaging (For examples, see Figure 1 j in reference Akram et al, 2019).


        Figure 3. MTT assay workflow. A. Allocation of PCLS for MTT assay. B. Plating of PCLS in 24-well plates for treating with MTT solution. C. PCLS are treated with MTT solution. D. Eluted formazan in wells of a 96-well plate.

    2. Live/Dead assay
      1. Perform cell viability assay on PCLS using LIVE/DEAD® Viability/Cytotoxicity Kit.
      2. Place four PCLS into an ibidi 24-well μ-plate for time-lapse imaging and four PCLS into a conventional incubator in a 24-well plate to culture for the same duration as the time-lapse without imaging, in 500 µl phenol red-free SF-DMEM. Place four PCLS into an ordinary 24-well plate to perform Live/Dead assay to evaluate viability at the initial time-point (0 h).
      3. For the non-imaged slices initial time-point (0 h), remove phenol red-free SF-DMEM and incubate PCLS with 2 µM Calcein AM and 2 µM Ethidium homodimer-1 (EthD-1) in 250 µl warm HBSS for 30 min at 37 °C, 1 PCLS per well.
      4. Wash the PCLS two times with HBSS and fix with 10% neutral buffered formalin for 30 min at room temperature (RT). Wash twice with HBSS and store the slices in HBSS at 4 °C prior to imaging.
      5. At the end of time-lapse imaging, remove transwells from each well as described for MTT assay and wash once with HBSS. Then perform Live/Dead assay on both time-lapse imaged and non-imaged PCLS, i.e., those cultured in conventional incubator for same duration as time-lapse, as above.
      6. As a positive control for dead cells, treat PCLS with 70% methanol for 30 min at RT and incubate with Calcein AM and EthD-1 as described above.
      7. Mount the PCLS on glass slides with a drop of ProLong® Gold Antifade Mountant and cover with a glass coverslip. Allow 1 h for mountant to set at room temperature.
      8. Capture images using a Zeiss LSM-510 confocal microscope with a 20x 0.8 NA air objective and ZEN 2009 (black edition) software (or equivalent).

  4. Cell tracking for cell migration quantification using Icy software
    1. Open the Icy software and import an EpCAM-FITC, SiR-DNA labeled raw time-lapse image sequence file.
    2. In the FITC channel, select the best focused plane, i.e., a single slice from the z-stack of the image sequence (Figure 4B).
    3. Select the desired time duration you wish to conduct cell tracking for (e.g., 8-16 h).
    4. Use the Spot detection and Tracking tools (under the Detection/Tracking tab) to detect EpCAM positive epithelial cells and track them throughout the pre-defined time-lapse duration.
    5. To detect EpCAM-FITC positive cells, set the Spot detector parameters as follows on full frame image: (a) Detect bright spot over dark background option, (b) Size of spots 7 and 13 pixels with 100% sensitivity, (c) Filtering parameter with 100-200 (adjust accordingly to correctly spot the individual cell), (d) Enable swimming pool option under the Output tab. Then Click ‘Start detection’. Check that the detector is identifying individual cells (which will be marked by red circle). If necessary correct spot detection by altering the Pixel size and Filtering parameters.
    6. After optimization of spot detection, click ‘Run Tracking’ to quantify the cell migration. Quantify cell migration on X-Y axis only.
      Note: 3D X-Y-Z axis migration quantification does not work accurately under this setting.
    7. After tracking, check every cell migration trail in each image field by rewinding the video sequence on Tracking mode a few times to see if there is any off-target tracking. If there is any off-target tracking, deselect these trails. Off-target tracking can occur when a trail is falsely generated without the presence of a migrating cell (Figure 4B).
      Note: This manual checking is crucial as under this setting Icy often tracks off-target objects. For cell tracking do not perform batch processing, it does not work under this setting. Track cells of each field from each PCLS individually slice by slice and by adjusting the detection parameters with post-tracking off-target screening for each video sequence.
    8. After completion of tracking add ‘Motion profiler processor’ to the ‘Track processor’ option under Track manager. Use Real unit option (µm/s) for migration and speed quantification from Track manager (Figure 4A).
    9. Export data as Excel file and the cell tracking trace as image file (Figures 4A and 4C).
      Note: For an example of generated cell tracking data, and cell migration data, see Figures 2 h, i, j in the reference Akram et al, 2019.


      Figure 4. Cell tracking by Icy. A. Raw cell tracking data from P3 PCLS generated by Icy Track Manager (22 out of 118 cell tracking data presented). B. Cell migration trails of 3 EpCAM-FITC positive cells from P3 PCLS tracked by Icy Track Manager showing 3 cells migrated from position 1 to position 2 in 16 h (zoomed raw image). Red arrows indicate the linear ‘Net cell migration’. C. Cell tracking trace produced from 1 full-frame visual filed from a P3 PCLS by Icy Track Manager.

  5. Image deconvolution and movie generation
    1. Image deconvolution using Huygens deconvolution software
      Note: The raw fluorescent images obtained by widefield microscopy from 300 µm thick PCLS are hazy and unsuitable for individual cell observations due to the limitations of widefield imaging, and light diffraction caused by sample thickness. Deconvolution software uses an algorithm to eliminate out-of-focus light and produce sharper images. (See Figure 1 i and Supplementary movie 2 in reference Akram et al., 2019).
      1. Upload the widefield z-stack of time-lapse fluorescent images to the Huygens deconvolution software (Step B2k). Each z-stack consists of 11 separate z-slices per sample, with a 1 µm step between slices.
        Note: The optimum step-gap between slices is dependent on the objective lens and requirements of the deconvolution software. For Huygens deconvolution software for 40x objective lens optimum step-gap is no more than 1 µm. This configuration may vary for other deconvolution software packages. Optimizing the step-gap is crucial to generate confocal-like, deconvolved images.
      2. Deconvolve the image sequence using the built-in automated CMLE algorithm with the following parameters: (a) Maximum iterations 50, (b) Signal to noise ratio 40, (c) Quality threshold 0.01, (d) Iteration mode Optimized.
      3. Save the deconvolved time-lapse image sequence as a TIFF z-stack image sequence file for 2D and 3D time-lapse video generation.
    2. 2D and 3D and bright-field video generation
      1. For 2D video generation, import deconvolved, time-lapse z-stacks into Icy software (Step E1c). Select the best-focused single z-plane, adjust the brightness and contrast and crop the area of interest. Save the file as .avi file format.
      2. For 3D video reconstruction, import deconvolved z-stack time-lapse files into NIS-Elements and align to correct for X-Y drift (Step E1c). Generate 3D videos using the ‘Volume View’ and ‘Movie Maker’ modules of NIS-Elements.
      3. For 2D brightfield video generation, import raw image z-tack files into Fiji (ImageJ) software. Select the best-focused single z-plane, adjust brightness and contrast. Save the file as .avi video file.
        Note: Brightfield images do not need deconvolution.
      4. For annotation on 2D fluorescent and brightfield videos, insert the .avi video files on Microsoft PowerPoint (MS Office 2016) as video file, resize the slide size to match with the video window dimensions, annotate the video accordingly using Text and Shape tools. Export the PowerPoint file as high resolution .mp4 movie file. (See supplementary movies in Akram et al., 2019).

Data analysis

  1. Quantification of cell behaviors
    1. To quantify cellular events, such as cell clustering, hollowing, septation and cell extension during post-natal alveologenesis in PCLS carefully examine deconvolved and brightfield time-lapse video files (using 40x objective) on screen. Record the number of times each event is observed in a single field of view from a video file. Examine videos from at least 4 different fields from different PCLS and from 3 independent experiments.
    2. Present data as the frequency of each type of cellular behavior as the total number of observations of that behavior per field. (For examples of results, see Results section, pages 4-7 in Akram et al., 2019)

  2. Quantification of cell migration
    1. Track manager in Icy produces a range of cell migration information from the tracking data including: Net cell migration, Total displacement, migration speed (Figure 4A). 
    2. To quantify and compare epithelial cell migration between different postnatal age groups (e.g., P3, P7, P14 and Adult) or different treatment conditions determine (a) Net cell migration and (b) the net distance traveled by individual cells within a specified time-period. Net cell migration is the mean value calculated to present how much linear distance in the X-Y axis (Initial point A to end point B) a cell migrates. A mean value of migrated distances of the total number of cells from each field is presented as mean net cell migration. (For example of results, see Figure 2 h, i, j in Akram et al., 2019).
      Note: For migration analysis use ‘Net Displacement’ not ‘Total Displacement’ data from cell tracking record (Figure 4A, yellow highlight).
    3. There are two groups of epithelial cells present in the lung slices: one group of cells are sessile, and other group of cells are highly motile. To differentiate these two groups and to compare them between different postnatal age groups and treatment conditions, determine the net distance traveled by individual cells. Rank them and present as percent or proportion of cells that migrate the ranked distances (See Figure 2 h-j and Figure 6 k-n in Akram et al., 2019).

Recipes

  1. HBSS/HEPES ice cold buffer
    1x HBSS
    1% HEPES 1 M
  2. Agarose solution
    1.5 g low-melting point agarose powder
    100 ml HBSS/HEPES buffer
    Dissolve agarose in HBSS/HEPES buffer in microwave. Prepare 30 ml agarose for processing 1 lung
  3. SF-DMEM
    DMEM basal media
    1% Penicillin-Streptomycin
  4. Image media
    Phenol red-free DMEM with HEPES basal media
    1% Penicillin-Streptomycin
    EpCAM-FITC antibody 1:500
    PECAM antibody 1:500 or SiR-DNA 1:1,000 
  5. MTT working solution
    DMEM basal media
    10% MTT stock solution (Stock Con. 5 mg/ml in DMSO)
    Note: Stock solution is stable for 1 year when stored at -20 °C in a dark container.
  6. 70% ethanol
    70 ml of absolute ethanol
    30 ml distilled water
  7. 70% methanol
    70 ml methanol
    30 ml PBS

Acknowledgments

This project was funded by a Leverhulme Trust grant to CHD (RPG-2015-226). The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part-supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and BBSRC (grant BB/L015129/1). This protocol is adapted from the method published in Akram et al., 2019.

Competing interests

The authors declare that they have no conflict of interest.

Ethics

All animal maintenance and procedures were carried out according to the requirements of the Animal (Scientific Procedures) Act 1986. Animal work was approved by the South Kensington and St. Mary’s AWERB committee, Imperial College London.

References

  1. Akram, K. M., Patel, N., Spiteri, M. A. and Forsyth, N. R. (2016). Lung regeneration: endogenous and exogenous stem cell mediated therapeutic approaches. Int J Mol Sci 17(1).
  2. Akram, K. M., Yates, L. L., Mongey, R., Rothery, S., Gaboriau, D. C. A., Sanderson, J., Hind, M., Griffiths, M. and Dean, C. H. (2019). Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour. Nat Commun 10(1): 1178.
  3. Amy, R. W., Bowes, D., Burri, P. H., Haines, J. and Thurlbeck, W. M. (1977). Postnatal growth of the mouse lung. J Anat 124(Pt 1): 131-151.
  4. Burgstaller, G., Vierkotten, S., Lindner, M., Konigshoff, M. and Eickelberg, O. (2015). Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue. Am J Physiol Lung Cell Mol Physiol 309(4): L323-332.
  5. Herriges, M. and Morrisey, E. E. (2014). Lung development: orchestrating the generation and regeneration of a complex organ. Development 141(3): 502-513.
  6. Hilgendorff, A., Reiss, I., Ehrhardt, H., Eickelberg, O. and Alvira, C. M. (2014). Chronic lung disease in the preterm infant. Lessons learned from animal models. Am J Respir Cell Mol Biol 50(2): 233-245.
  7. Hind, M., Corcoran, J. and Maden, M. (2002). Alveolar proliferation, retinoid synthesizing enzymes, and endogenous retinoids in the postnatal mouse lung. Different roles for Aldh-1 and Raldh-2. Am J Respir Cell Mol Biol 26(1): 67-73.
  8. Kotton, D. N. and Morrisey, E. E. (2014). Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat Med 20(8): 822-832.
  9. Kreiger, P. A., Ruchelli, E. D., Mahboubi, S., Hedrick, H., Scott Adzick, N. and Russo, P. A. (2006). Fetal pulmonary malformations: defining histopathology. Am J Surg Pathol 30(5): 643-649.
  10. Li, J., Wang, Z., Chu, Q., Jiang, K., Li, J. and Tang, N. (2018). The strength of mechanical forces determines the differentiation of alveolar epithelial cells. Dev Cell 44(3): 297-312 e5.
  11. Madurga, A., Mizikova, I., Ruiz-Camp, J. and Morty, R. E. (2013). Recent advances in late lung development and the pathogenesis of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 305(12): L893-905.
  12. McGowan, S. E. (2014). Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation. Birth Defects Res A Clin Mol Teratol 100(3): 227-239.
  13. Meng, Q. J., McMaster, A., Beesley, S., Lu, W. Q., Gibbs, J., Parks, D., Collins, J., Farrow, S., Donn, R., Ray, D. and Loudon, A. (2008). Ligand modulation of REV-ERBalpha function resets the peripheral circadian clock in a phasic manner. J Cell Sci 121(Pt 21): 3629-3635.
  14. Mund, S. I., Stampanoni, M. and Schittny, J. C. (2008). Developmental alveolarization of the mouse lung. Dev Dyn 237(8): 2108-2116.
  15. Pieretti, A. C., Ahmed, A. M., Roberts, J. D., Jr. and Kelleher, C. M. (2014). A novel in vitro model to study alveologenesis. Am J Respir Cell Mol Biol 50(2): 459-469.
  16. Poobalasingam, T., Salman, D., Li, H., Alcada, J. and Dean, C. H. (2017). Imaging the lung: the old ways and the new. Histol Histopathol 32(4): 325-337.
  17. Roth-Kleiner, M. and Post, M. (2005). Similarities and dissimilarities of branching and septation during lung development. Pediatr Pulmonol 40(2): 113-134.
  18. Sanderson, M. J. (2011). Exploring lung physiology in health and disease with lung slices. Pulm Pharmacol Ther 24(5): 452-465.
  19. Snoeck, H. W. (2015). Modeling human lung development and disease using pluripotent stem cells. Development 142(1): 13-16.
  20. Thornton, E. E., Krummel, M. F. and Looney, M. R. (2012). Live imaging of the lung. Curr Protoc Cytom Chapter 12: Unit12 28.
  21. Warburton, D., Gauldie, J., Bellusci, S. and Shi, W. (2006). Lung development and susceptibility to chronic obstructive pulmonary disease. Proc Am Thorac Soc 3(8): 668-672.

简介

肺泡是肺的气体交换单位。肺泡发育,肺泡生成的过程受信号通路的复杂网络调节,该信号通路作用于各种细胞类型,包括I型和II型肺泡上皮细胞,成纤维细胞和血管内皮。肺泡生成失调导致新生儿和成人支气管肺发育不良,肺泡再生受阻与慢性肺部疾病(包括COPD和肺纤维化)有关。因此,可视化肺泡形成对于了解肺稳态和对于开发不可治愈的肺部疾病的有效疗法至关重要。我们已经开发出一种技术,可以结合使用宽视野显微镜和精确切割的肺切片的图像去卷积实时可视化肺泡形成。在这里,我们将详细介绍这种实时成像技术。这种延时成像技术可用于在较长时间段(长达16小时)内捕获组织切片内单个细胞的动态,而荧光或细胞毒性的损失最小。
【背景】 产前和产后肺发育分为几个不同的阶段,从前肠内胚层出芽开始,然后是周围肺间充质的分支形态发生,囊化和肺泡形成。这个发育过程受到精心安排的信号传导程序和细胞成分的严格调控(Kotton和Morrisey,2014年; Akram等人,2016年)。肺的主要功能是气体交换,这是通过扩散发生的(Herriges和Morrisey,2014年)。这种扩散发生在肺泡上皮和毛细血管内皮细胞薄层之间(Roth-Kleiner和Post,2005)。肺泡生成失调与许多新生儿和婴儿疾病有关,包括支气管肺发育不良(BPD)和肺发育不良(Kreiger等,2006; Hilgendorff等,2014)。 )。在成年人中,肺泡损伤是几种慢性肺部疾病的一部分,例如慢性阻塞性肺疾病(COPD)和特发性肺纤维化(IPF)。目前,除了肺移植以外,没有针对这些疾病的治疗方法(Warburton等,2006; Madurga等,2013; McGowan,2014),并且有为了开发有效的治疗方法,尚未理解肺泡形成的机制。
在小鼠中,接种开始于胚胎第17.5天左右,随后发生肺泡形成,该过程在出生后的头几天开始,并且大部分在出生后的第一个月内完成(Herriges和Morrisey,2014)。然而,肺泡生成最活跃的阶段发生在产后生命的前两周,大部分肺泡是在出生后第21天形成的(Hind et al。,2002; Snoeck,2015)。

根据静态成像实验,目前的认识是,肺泡的发生是通过反复的隔壁事件而发生的,这些事件将初级气囊细分以增加肺泡的数量和表面积(Amy et al。,1977; Mund <等等。,2008)。由于其在体内的深处以及相对较慢的持续时间,因此实时可视化肺泡形成具有挑战性。一项最近的研究同时使用了“体外”和“体内”实时成像技术来研究肺泡生成之前小鼠肺发育的接种阶段,但是这些技术不适用于成像产后肺(Poobalasingam等,2017; Li等,2018)。
精确切割的肺切片(PCLS)包含完整的肺泡,并越来越多地用于研究肺生物学和疾病的发病机制(Meng等,2008; Sanderson,2011; Thornton等,,2012年)。 PCLS的延时成像已用于显示成年正常和纤维化小鼠肺以及人肺PCLS中间充质细胞和巨噬细胞与细胞外基质的动态相互作用(Burgstaller等人。 ,2015)。此外,在出生后的小鼠早期PCLS培养物中已证明了可量化的体外肺泡形成(Pieretti et al。,2014)。结合宽视野显微镜和图像反褶积技术对出生后小鼠PCLS的研究,我们开发了一种实时捕获肺泡形成形态机制的方法(Akram et al。,2019)。在这里,我们描述了产后肺泡生成实时实时成像的详细协议。

关键字:肺泡生成成像, 延时成像, 反卷积软件, 离体肺组织切片, 肺组织发育

材料和试剂

  1. 50 ml离心管(Thermo Fisher,目录号:338652)
  2. 金属锅铲(Fisher Scientific,目录号:11523482)
  3. 玻璃盖玻片(Thermo Fisher,货号:102260)
  4. 用于P3小鼠的25 G,19 mm(0.75英寸)的Probe Point(Blunt)针(哈佛仪器,目录号:725461);带有铝制轮毂的Monoject钝针,用于P7小鼠为23 G,1英寸(哈佛仪器,目录号:722349),用于P14和成年小鼠的21 G(由21 G注射器针头定制)(均来自英国Harvard Apparatus)
  5. 24孔板(Corning ® Costar ® TC处理的多孔板)(Sigma-Aldrich,CLS3527-100EA)
  6. 96孔板(Thermo Fisher,货号:249952)
  7. 伊比迪(Ibidi)24孔μ板(未涂覆)(伊比迪(Ibidi),目录号:82401)
  8. Transwell(0.4 µm孔,12 mm,聚酯膜)(Corning,目录号:3460)
  9. 显微镜载玻片,SuperFrost ®,MenzelGläser(VWR,目录号:631-1318)
  10. 非无菌丝绸黑色编织缝合线轴,22.9 m,尺寸5-0(英国哈佛仪器公司,目录号:517607)
  11. BD Micro-Fine + 29 G,1 ml胰岛素注射器(MediSuuplies,目录号:PMC3743)
  12. 5毫升注射器(VWR International,目录号:SART16644-E)
  13. Thermo Scientific TM Nunc TM 细胞培养/培养皿,100毫米(Fisher Scientific,目录号:10508921)
  14. Swann-Morton手术刀,第22号(MediSupplies,目录号:PMC0105)
  15. 湿冰和冰盒
  16. 查尔斯河实验室的C57BL / 6雄性和雌性小鼠
  17. EpCAM-FITC(CD326)单克隆抗体(eBioscience,目录号:11-5791-80;克隆G8.8)
  18. Alexa-647偶联的PECAM抗体(CD31-Alexa 647)(Biolegend,目录号:102416;克隆390)
  19. 实验室薄页纸(蓝色卷)
  20. 金属平垫圈(重量1.66克)(M8-5 / 16 th 英寸直径)(英国B&amp; Q)
  21. 绝对乙醇(Sigma-Aldrich,目录号:34852-M)
  22. 戊巴比妥(Pentoject,Animalcare,目录号:XVD 132)
  23. 低熔点琼脂糖(Sigma-Aldrich,目录号:A9414)
  24. 汉克斯平衡盐溶液(HBSS)(1x)(Life Technologies,目录号:14025-050)
  25. HEPES 1 M(Life Technologies,Gibco,目录号:15630080)
  26. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco,目录号:20012068)
  27. Dulbecco的改良Eagle培养基(DMEM)(Life Technologies,Gibco,目录号:31966-021),在食谱中也称为“ DMEM基础培养基”
  28. 青霉素-链霉素(10,000 U / ml)(Life Technologies,Gibco,目录号:15140122)
  29. MTT试剂(噻唑基溴化四氮唑蓝)(Sigma-Aldrich,目录号:M2128)
  30. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:276855)
  31. 10%中性福尔马林缓冲液(Sigma-Aldrich,目录号:HT501128)
  32. DAPI(原料浓度10 mg / ml)(Sigma-Aldrich,目录号:D9542)
  33. LIVE / DEAD ®活力/细胞毒性试剂盒(Thermo Fisher Scientific,目录号:L3224)
  34. 甲醇(Sigma-Aldrich,目录号:34860)
  35. ProLong ®防锈金贴剂(Thermo Fisher Scientific,目录号:P36930)
  36. 罗丹明硅远红外荧光团缀合的DNA小沟结合剂双苯甲酰亚胺(SiR-DNA)(tebu-bio Ltd,目录号:SC007)
  37. 配方中含有HEPES的不含酚红DMEM(生命技术,目录号:21063029),在配方中也称为“含HEPES基础培养基的不含酚红DMEM”
  38. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)
  39. 海卫一X-100(Sigma-Aldrich,目录号:X100)
  40. (可选)对硝基硝基苄青霉素(开曼化学公司,目录号:13891)
  41. (可选)细胞松弛素D(Sigma-Aldrich,目录号:C8273)
  42. HBSS / HEPES冰冷缓冲液(请参阅食谱)
  43. 琼脂糖溶液(请参阅食谱)
  44. SF-DMEM(请参阅食谱)
  45. 图像媒体(请参阅食谱)
  46. MTT工作解决方案(请参阅食谱)
  47. 70%乙醇(请参阅食谱)
  48. 70%甲醇(请参阅食谱)

设备

  1. 弯曲解剖钳,10厘米,Serr / C(世界精密仪器有限公司,目录号:15915)
  2. 细尖解剖剪刀,直,10厘米(World Precision Instruments Ltd,目录号:14393)
  3. 14厘米直形手术剪刀(World Precision Instruments Ltd,目录号:14192)
  4. 弹簧剪刀,直12厘米,超细刀片12毫米(World Precision Instruments Ltd,目录号:14125)
  5. 用于振动刀的不锈钢刀片(Campden Instruments LTD,目录号:7550-1-SS)
  6. 自动化振动刀(Compresstome ® VF-300-0Z;美国精密仪器有限责任公司)
  7. 保温箱(加湿器,37°C,5%CO 2 )
  8. -20°C冷冻室
  9. GFP滤光片,激发450-490 nm,发射500-550 nm(用于EpCAM-FITC)
  10. Cy-5滤光片,激发625-655 nm,发射665-715 nm(用于SiR-DNA和PECAM)
  11. 酶标仪(Tecan; Sunrise TM ,INSTSUN-1)
  12. 蔡司Axio Observer倒置宽视野显微镜,配备Lumencor Spectra X LED光源和Hamamatsu Flash 4.0相机(德国蔡司)
  13. 蔡司LSM-510倒置共焦显微镜(德国蔡司,型号:LSM 510)
  14. Leica DM2500宽视野显微镜(Leica Microsystems,型号:Leica DM2500)

软件

  1. Zen2采集软件,蓝色版本(德国蔡司)
  2. ZEN 2009(黑色版)软件(德国蔡司)
  3. FIJI(ImageJ,版本2.0)
  4. 冰冷的开源生物成像分析软件(1.9.8.0版;由法国巴黎巴斯德研究所的定量图像分析部门创建)
  5. 惠更斯反卷积软件(Scientific Volume Imaging,SVI,Essential版本17.10)
  6. NIS-Elements(4.50版,英国尼康仪器公司)
  7. GraphPad Prism版本5
  8. Microsoft Excel(Microsoft Office 2011版本)
  9. Microsoft PowerPoint(Microsoft Office 2016版本)

程序

  1. 生产精确切割的肺片
    1. 小鼠解剖和肺收获
      1. 通过腹膜内注射戊巴比妥(50 mg / kg体重),人道地杀死出生后第3天(P3),P7,P14或成年(6-8周)C57BL / 6小鼠。使用29 G,1 ml胰岛素注射器注射25μlP3戊巴比妥,P7 50μl,P14 75μl和成年小鼠100μl(图1A7)。注射后5至10分钟内小鼠会死亡。通过对股动脉放血确认死亡。
      2. 将鼠标仰卧在解剖盘上。在腹部,胸部和颈部的前部喷洒70%的乙醇。
      3. 用弯曲的镊子(图1A2)从前腹壁捏住皮肤,然后轻轻向上拉,切开一小块皮肤,使内腹膜完好无损。
      4. 使用一把细剪刀(图1A4)沿前腹部和胸壁的中线做一个纵向切口,并将其延伸到下巴(图1B)。
      5. 使用两对弯曲的镊子,使两侧的皮肤缩回,露出腹部,胸壁和颈部的前侧。 (图1Ci)(视频1)。
      6. 用弯曲的镊子小心地提起腹部上部腹膜壁,正好位于肋缘以下,并用细剪刀的尖端开一个小开口。
      7. 用细剪刀将开口向左右延伸。这将使隔膜露出。
      8. 轻轻抬起胸骨的下端,并用细剪刀的尖端在隔膜的上部开口。然后将开口沿肋缘的内表面延伸到隔膜的两侧(图1C i,ii,黄色虚线)。在此阶段,您将能够看到浅粉红色的左右折叠肺(图1C iii,箭头)。
      9. 用弯曲的镊子握住胸骨的下端提起胸壁,并小心地做胸骨旁切口(向左和向右),使前胸壁与人体分离(图1C i,iii,黄色虚线)(视频1 )。
      10. 使用弯曲的镊子和细剪刀,从颈部壁的前侧和外侧去除肌肉和筋膜。
      11. 使用弯曲的镊子,在颈部中点在气管后侧和食道前壁之间开一个开口(视频1)。
      12. 通过开口插入一块无菌缝合线,并将其垂直于气管的中点放置(图1)。
      13. 使用细尖的直形弹簧剪刀在环状软骨下方的气管前壁上开一个小开口(图1A,(6),1C(iii)黄色箭头,图1D(ii)插图)。
      14. 小心地将硬质金属插管25 G(用于P3),23 G(用于P7)和21 G(用于P14和成年小鼠)穿过气管,直到主支气管分叉处高出一毫米,然后用缝合线固定到位(图1C(iv)和一维(ii)插图)。通过施加两个结,将缝合线以适度的紧度绑在气管周围,包括内部的插管。
      15. 插管后,轻轻注入1.5%的低熔点琼脂糖,使之膨胀,该琼脂糖应加温至37°C的HBSS / HEPES缓冲液(请参见食谱)。使用5毫升注射器注入琼脂糖,并将注射器连接到套管,直到琼脂糖固化。给两个肺充气,使它们保持在胸腔内原位,使用P3为0.2 ml,P7为0.275 ml,P14为0.35 ml和成人肺为1 ml。这些体积使肺部完全膨胀,而不会出现过度或次佳的膨胀(图1C和1D)。
      16. 充气后,在裸露的胸壁上放一块双层实验室蓝色卷,然后在蓝色卷上放一些湿冰,使琼脂糖固化。
        注意:根据鼠标的年龄,这将需要1-2分钟。
      17. 从气管中轻轻拔出套管,然后拧紧缝合线结。
      18. 从其上端(嘴)将气管放出来。握住缝合线的自由端提起气管,并从胸腔中轻轻移走整个心脏和肺部(图1C和1D)。
      19. 将整个心脏和肺部浸入50毫升离心管中的无冰冷无血清DMEM(SF-DMEM)中,盖紧盖子并保存在湿冰上直至切片。组织可以在冰上放置2-3小时。


        视频1.鼠标解剖:从皮肤切口到气管动员


        图1.解剖小鼠和收集肺部的工作流程。 A.解剖设备:1.缝合线2.弯曲钳3.尖端镊子4.尖端解剖剪刀5.手术剪刀6 。弹簧剪刀,7.注射皮下注射针的1 ml注射器中的戊巴比妥。 B.皮肤切口线。 C.成年小鼠解剖,琼脂糖膨胀和肺部收获。 D.产后第3天(P3)小鼠解剖,琼脂糖充盈和肺部收获。

    2. 精确切肺切片
      1. 准备自动振动刀:通过喷雾70%乙醇彻底消毒振动刀的缓冲液托盘,然后晾干(图2A)。使用强力胶将振刀的刀片固定在刀片支架上,然后晾干。使用70%乙醇喷雾消毒样品架和样品管并风干。在使用前,将注射器冷却块放在-20°C的冰箱中15-30分钟以使其冷却(图2B)。
      2. 准备在37°C的温度下用HBSS / HEPES制备约30 ml的1.5%琼脂糖凝胶。准备200 ml无菌冰冷的1x HBSS / HEPES缓冲液,并保持在冰上。用每孔1 ml冰冷的无菌SF-DMEM制备24孔板,并保持在冰上。
      3. 在直径为10 cm的培养皿中,用手术刀刀片将左肺叶与左支气管分开(图2C和2D)。
      4. 使用手术刀刀片从肺根部切除一小部分组织(图2D;切除的组织轮廓用虚线标记)。
        注意:这有助于将瓣垂直放置在样品架上。
      5. 将肺叶放在一块干净的实验室蓝色卷上,以吸收多余的介质(图2D)。
      6. 将一滴超级胶水滴在样品架的切割端上(图2Ei),然后将肺叶的切割端轻轻地放在胶滴上,用弯曲的镊子将其垂直固定,直到肺叶牢固地粘附在样品架上(图2Eii)。
        注意:将肺叶附着在标本支架上需要15到30 s。
      7. 将标本支架插入金属标本管,垂直握住,使肺叶保持在标本管内并垂直面向上(图2Eiii)。
      8. 使用5 ml注射器,将37°C的1.5%温暖的琼脂糖填充到样品管中,以使整个叶浸没在琼脂糖中(图2Eiii)。
      9. 将冷注射器冷却块(图2B)放在样品管周围,并将其固定到位,直到琼脂糖组织块凝固(图2Eiv)。
        注意:凝固琼脂糖大约需要一分钟。
      10. 将标本管插入缓冲液托盘,然后将刀片和琼脂糖组织块的切割端对齐。
      11. 用冰冷的HBSS / HEPES缓冲液填充缓冲液托盘。将所需的组织切片厚度设置为300 µm。将切割速度设置为5,将切割振荡频率设置为5。在“连续模式”下操作振动刀。
      12. 开始收集横向精确切割的肺切片(PCLS)。使用小金属刮刀从缓冲液托盘中取出肺叶的前1/3 rd 个肺片。从叶的中间1/3 rd 收集切片,并将其放在装有冰冷SF-DMEM的24孔板的孔中(图2F)。肺叶的中间三分之一提供几乎相等大小的切片。在每孔1 ml培养基中取1个肺切片。 (使用此技术,P3左肺提供12个切片,成年肺提供大约36个切片。)
        注意:使用相等大小的切片进行成像和代谢测定非常重要,其余的切片可用于其他测定,例如 例如 ,免疫染色,蛋白质或RNA提取。
      13. 在5%CO 2 存在下于37°C孵育PCLS 2 h,然后在无菌条件下,在通风橱中,用温热SF-DMEM洗涤两次以除去组织周围多余的琼脂糖。
        注意:一些琼脂糖会残留在肺部的气隙中,这对于在培养过程中保持肺部结构很重要。
      14. 在5%CO 2 存在下,将PCLS在SF-DMEM中于37°C孵育1小时。至此,PCLS已准备好进行实时成像和进一步的实验。
        注意:立即将样品用于成像(和迁移分析);但是切片可以过夜培养,以进行其他实验。

  2. 肺泡发生的活细胞成像
    1. 无标签的明场实时成像
      为了可视化肺切片随时间变化的总体结构变化,可以按以下方式执行实时明场成像:
      1. 使用不含酚红的DMEM和ibidi 24孔μ板进行实时成像。
      2. 准备用于成像的Transwell:使用剪刀剪下并去除Transwell的边缘,以使Transwell过滤器与孔底部的肺切片接触(图2G)。用HBSS(温热)清洗跨孔,以除去碎屑,并将其放置在没有缓冲液的ibidi 24孔μ板的未使用孔中。
      3. 将50 µl温暖(37°C)的无酚红SF-DMEM加入ibidiμ平板孔的中心。
      4. 使用薄的无菌金属刮刀从上面的步骤A2n中取出PCLS,然后小心地将其放置在ibidiμ平板孔的中心。确保将肺片放在中间并且平坦且没有折叠。
      5. 然后将修饰的transwell轻轻放在PCLS的顶部。
      6. 将500 µl不含酚红的SF-DMEM加入到Transwell的上腔室中,并将300 µl加入到Transwell的下腔室中。
      7. 为了将PCLS固定在适当的位置,请在Transwell外壳顶部放置一个带有中心孔的1.66 g金属平垫圈(图2H和2I)。
        注意:此重量通过可渗透的Transwell膜在组织切片上施加轻微压力,该膜将切片固定在适当的位置,而不会损坏组织。金属平垫圈的重量不应超过3.32克。
      8. 将PCLS在37°C下,在5%CO 2 和95%空气(〜21%O 2 )存在的潮湿培养箱中孵育2小时。此温育步骤使肺切片在图像采集之前安定下来。
        注意:实时图像后,应分配大小相似的PCLS进行MTT活力测定,如图3所示。
      9. 同时,在以下条件下(37°C,5%CO 2 )加湿并预先平衡Zeiss Axio Observer宽视野落射荧光显微镜(或类似设备)的培养箱,持续30分钟至1小时。 sub>和室内空气中的氧气水平,大约21%。
      10. 孵育和预平衡后,将包含PCLS的ibidi 24孔板转移至显微镜培养箱,并在整个延时过程中保持上述培养箱条件。
      11. 使用长工作距离40倍(0.7 NA,空气)物镜在强光下开始捕获延时图像(图2I)。
      12. 对于明场成像,请在感兴趣的区域找到最佳聚焦平面,并保持该点为中心,以1 µm的步长选择朝向切片上表面的30个z切片和朝向切片底部表面的30个切片,切片之间的间隙(在组织的60 µm z轴中间总共有60个切片)。
      13. 将延时拍摄图像间隔设置为每15分钟12-19小时。
        注意:每小时四张图像(间隔15分钟)减少了曝光量,从而最大程度地减少了荧光团漂白现象,但可以产生10-12fps的平滑延时视频(请参阅Akram等人的补充电影2,2019)。
      14. 从PCLS的四个象限中选择4个单独的感兴趣的区域,避开气道,进入肺泡区域。
        注意:显微镜培养箱的2µh孵育和预平衡对于在延时图像采集过程中避免焦点平面漂移至关重要。图片每个实验4个单独的PCLS。


        图2.精确切割的肺切片和实时成像设置。 A.肺切片机:Vibratome,Compresstome ® VF-300-0Z。 B.用于凝固琼脂糖的注射器冷却块。 C.从P3小鼠收获的琼脂糖使肺膨胀。 D.在实验室组织卷上分离左肺叶。 E.显示左肺叶琼脂糖包埋在用于制备PCLS的标本支架上的示意图。 F.精确切割的肺切片在24孔板中。 G.修饰transwell以稳定孔板上的肺切片。 H.精确切割的肺切片使用改良的transwell和金属平垫圈(黑色边框)设置在ibidi 24孔μ板上。 I.示意图显示了倒置显微镜上的金属平垫圈,transwell,肺切片和40倍长工作距离空气物镜的相对位置和对准,用于实时成像。

    2. 免疫染色PCLS的实时荧光成像
      用于实时成像的PCLS双重染色
      1. 为了可视化和跟踪PCLS中的上皮细胞,请使用结合FITC的EpCAM抗体(EpCAM-FITC)和SiR-DNA对双色细胞进行染色。 (EpCAM选择性标记上皮细胞膜。SiR-DNA是若丹明硅红光荧光团缀合的DNA小沟结合剂双苯甲酰亚胺,可染色细胞核)。
      2. 将PCLS(来自步骤A2n)在37°C下与1:200稀释的EpCAM-FITC抗体和1:300稀释的SiR-DNA在DMEM中孵育(不振摇)。每个PCLS每孔使用500μl抗体/ SF-DMEM培养基。
      3. 为了观察肺泡区域内的上皮细胞和内皮细胞,将PCLS与Alexa-647偶联的PECAM抗体和EpCAM-FITC抗体在37°C下分别以1:200在每孔每个样品500 µl SF-DMEM中孵育1 h。
      4. 孵育后,用温热的SF-DMEM清洗PCLS 3次。

      PCLS的延时荧光成像
      1. 图像介质:通过添加1:500的EpCAM-FITC抗体和1:1000的SiR-DNA用于上皮细胞或添加1:500的PECAM以及EpCAM-FITC(1:500)进行双标记的上皮/每个孔中每个PCLS含800 µl无酚红SF-DMEM的内皮细胞。
        注意:1:1000稀释的SiR-DNA可用作上皮或内皮标记双重标记的细胞的核标记。
      2. 将50 µl图像介质滴放在ibidi 24孔μ板的孔中心,并用薄金属刮刀小心地将双重染色的PCLS放在图像介质上。
      3. 将改良的Transwell放置在PCLS的顶部,将500 µl图像介质添加到上腔室中,并将300 µl图像介质添加到底部腔室中,最后通过如上所述将平坦的金属垫圈放在Transwell上来稳定Transwell。 2I)。
      4. 将PCLS在5%CO 2 和95%空气存在下于37°C的潮湿培养箱中培养2小时。此孵育步骤会增强染色效果,并允许肺片在图像采集之前安定下来。
      5. 按照用于明场成像的说明预先平衡显微镜培养箱(如步骤B1i)。
      6. 孵育和预平衡后,将24孔板转移至倒置的Zeiss Axio Observer宽视野落射荧光显微镜培养箱中,并在整个延时时间内保持上述培养箱条件。
      7. 使用GFP滤光片,使用长工作距离40倍(0.7 NA,空气)物镜开始记录延时图像,激发450-490 nm,发射500-550 nm(对于EpCAM-FITC)和Cy-5,激发625- 655 nm,每片从肺泡区域的4个场发射665-715 nm(用于SiR-DNA和PECAM),持续15-19分钟,持续8-19 h。沿z轴从中间11 µm厚度的组织中捕获11张图像,每个切片之间有1 µm的步距,以从每个PCLS组成z堆栈。 注意:
        1. 每个实验一次在四个PCLS上进行延时成像。不要在实验中从同一PCLS捕获明场图像和荧光图像。暴露于强光下会大大削弱荧光染色的强度。请勿使用UV或DAPI滤光片(激发358 nm,发射463 nm)对PCLS进行实时成像,因为这会导致大量细胞死亡。 Zen2采集软件,蓝色版本用于明场和荧光实时成像。
        2. 在活细胞成像过程中,可以通过向图像介质中添加各种因子(例如细胞因子或小分子抑制剂)来操纵细胞动力学或其他行为。所产生的细胞反应可以实时记录。例如,我们最近在PCLS中添加了肌动蛋白细胞骨架修饰剂Blebbistatin和Cychachalasin-D,并使用这种live-PCLS成像技术评估了它们对细胞迁移的影响(Akram et al。 ,2019)。
        3. 为确保PCLS的生存力,请在每次延时实时成像实验后对PCLS进行生存力分析。对于未标记的PCLS上的明场成像,请进行活/死染色;对于双重染色的PCLS上的荧光成像,请进行MTT分析。
        4. 实验的持续时间p:从解剖小鼠到延时成像的开始需要7-8小时。

  3. 活细胞成像实验后在PCLS上进行细胞活力测定
    1. MTT测定
      1. 将四个已经过实时成像的PCLS置于ibidi 24孔μ平板中,每孔1片。将四个相似大小的未成像PCLS放置在37°C,5%CO 2 的培养箱中,放置时间与传统24孔板中成像的切片相同,持续时间为1切片每孔加入无酚红SF-DMEM。选择四个相似大小的切片进行MTT分析,以在延时成像实验开始时确定细胞活力(图3)。
      2. 为了评估在实时成像(0小时控制)开始时细胞内的代谢活性,向每个孔中添加500μlSF-DMEM制成的10%MTT溶液(Stock Con。5 mg / ml)。
        注意:此MTT必须在开始实时成像之前完成(图3B)。
      3. 将PCLS在5%CO 2 存在下于37°C孵育1小时(图3C)。
      4. 从孔中取出MTT介质。
      5. 加入等体积的DMSO(500 µl),并在37°C下孵育10分钟,以溶解活细胞内形成的甲maz晶体。
      6. 从每个PCLS中取出200 µl洗脱的甲maz溶液,并将其放入96孔板的各个孔中(图3D)。
      7. 在570 nm下测量吸光度(OD),并使用酶标仪在690 nm下校正。
      8. 要评估延时PCLS内的代谢活性,请在延时成像完成后小心移开transwell。如果PCLS粘附在Transwell PET膜的下表面,请用移液管用温暖的HBSS冲洗,以使其脱离。
      9. 用温暖的HBSS清洗PCLS一次。
      10. 然后,以与上述初始时间点控制PCLS相同的方式,对在常规培养箱中培养的经过延时成像和未成像的PCLS进行MTT分析。
      11. 比较初始时间点(0 h)和延时后(成像和未成像的PCLS)OD值,以评估延时成像后的细胞活力(例如,参见参考Akram等人的图1j等,2019) 。


        图3. MTT分析工作流程。 A.为MTT分析分配PCLS。 B.将PCLS镀在24孔板中,用MTT溶液处理。 C. PCLS用MTT溶液处理。 D.在96孔板的孔中洗脱甲maz。

    2. 活/死分析
      1. 使用LIVE / DEAD ®活力/细胞毒性试剂盒在PCLS上进行细胞活力测定。
      2. 将四个PCLS放入ibidi 24孔μ板中进行延时成像,将四个PCLS放入24孔板中的常规培养箱中,以与不进行成像的延时相同的时间在500 µl酚红中培养。免费的SF-DMEM。将四个PCLS放入普通的24孔板中,进行活/死分析,以评估在初始时间点(0 h)的生存力。
      3. 对于未成像的切片的初始时间点(0 h),除去不含酚红的SF-DMEM,并将PCLS与2 µM Calcein AM和2 µM Ethidium homodimer-1(EthD-1)在250 µl温暖的HBSS中孵育30分钟在37°C下每分钟1 PCLS。
      4. 用HBSS洗涤PCLS两次,并在室温(RT)下用10%中性福尔马林缓冲液固定30分钟。用HBSS清洗两次,并将切片在成像前于4°C的HBSS中保存。
      5. 在延时成像结束时,按照MTT分析所述,从每个孔中移出transwell,并用HBSS清洗一次。然后,对经过延时成像和未成像的PCLS(即)进行活/死检测,如上所述,这些PCLS是在常规培养箱中培养与延时相同的持续时间。
      6. 作为死细胞的阳性对照,如上所述,用70%甲醇在室温下处理PCLS 30分钟,然后与钙黄绿素AM和EthD-1一起孵育。
      7. 用一滴ProLong ®黄金防褪色安装剂将PCLS安装在载玻片上,并盖上玻璃盖玻片。让封固剂在室温下放置1小时。
      8. 使用配备20x 0.8 NA空气物镜的Zeiss LSM-510共焦显微镜和ZEN 2009(黑色版)软件(或同等软件)捕获图像。

  4. 使用Icy软件进行细胞跟踪以进行细胞迁移定量
    1. 打开Icy软件,然后导入EpCAM-FITC,SiR-DNA标记的原始延时图像序列文件。
    2. 在FITC通道中,选择最佳聚焦平面即,它是图像序列z堆栈中的单个切片(图4B)。
    3. 选择您希望进行电池跟踪的所需时间(例如 8-16小时)。
    4. 使用“斑点检测和跟踪”工具(在“检测/跟踪”选项卡下)来检测EpCAM阳性上皮细胞,并在预定义的时间间隔内跟踪它们。
    5. 要检测EpCAM-FITC阳性细胞,请在全帧图像上按以下步骤设置点检测器参数:(a)在深色背景上检测亮点选项;(b)以100%的灵敏度检测7和13像素点的大小;(c)过滤参数设置为100-200(进行相应调整以正确定位单个单元格),(d)在“输出”选项卡下启用游泳池选项。然后点击“开始检测”。检查检测器是否正在识别单个细胞(将用红色圆圈标记)。如有必要,可以通过更改像素大小和过滤参数来正确检测斑点。
    6. 优化斑点检测后,单击“运行跟踪”以量化细胞迁移。仅量化细胞在X-Y轴上的迁移。
      注意:在此设置下3D X-Y-Z轴迁移量化无法正确进行。
    7. 跟踪之后,通过在跟踪模式下倒回视频序列几次以检查每个图像字段中的每个单元迁移轨迹,以查看是否存在脱靶跟踪。如果存在任何偏离目标的跟踪,请取消选择这些跟踪。如果在不存在迁移单元的情况下错误生成了跟踪,则会发生脱目标跟踪(图4B)。
      注意:手动检查至关重要,因为在此设置下,Icy经常跟踪脱靶对象。对于单元格跟踪,请不要执行批处理,它在此设置下不起作用。通过逐个片段地逐个片段地跟踪每个PCLS的每个场的细胞,并通过跟踪每个视频序列的脱靶后筛选来调整检测参数。
    8. 跟踪完成后,在“跟踪管理器”下的“跟踪处理器”选项中添加“运动分析器处理器”。使用真实单位选项(µm / s)从Track Manager进行迁移和速度量化(图4A)。
    9. 将数据导出为Excel文件,并将单元格跟踪跟踪导出为图像文件(图4A和4C)。
      注意:有关生成的细胞跟踪数据和细胞迁移数据的示例,请参见参考文献Akram等人于2019年的图2 h,i,j。


      图4.通过Icy进行单元跟踪。 A.由Icy Track Manager生成的来自P3 PCLS的原始单元跟踪数据(显示的118个单元跟踪数据中的22个)。 B. Icy Track Manager跟踪的来自P3 PCLS的3个EpCAM-FITC阳性细胞的细胞迁移轨迹,由Icy Track Manager跟踪,显示3个细胞在16小时内从位置1迁移到位置2(原始图像放大)。红色箭头表示线性的“净细胞迁移”。 C.由Icy Track Manager从P3 PCLS归档的1个全帧视觉文件产生的细胞跟踪轨迹。

  5. 图像反卷积和电影生成
    1. 使用惠更斯反卷积软件进行图像反卷积
      注意:由于宽视野成像的限制以及样品厚度引起的光衍射,通过宽视野显微镜从300 µm厚的PCLS上获得的原始荧光图像模糊且不适合单个细胞观察。反卷积软件使用一种算法来消除散焦光并产生更清晰的图像。 (请参见参考Akram 等人 ,2019年的图1i和补充电影2)。
      1. 将延时荧光图像的广域z堆栈上传到惠更斯反卷积软件(步骤B2k)。每个z堆栈由每个样本11个独立的z切片组成,每个切片之间的间距为1 µm。
        注意:切片之间的最佳步距取决于物镜和解卷积软件的要求。对于用于40倍物镜的惠更斯反卷积软件,最佳步距不超过1 µm。对于其他解卷积软件包,此配置可能有所不同。优化阶跃间隙对于生成类似共焦的,反卷积的图像至关重要。
      2. 使用具有以下参数的内置自动CMLE算法对图像序列进行反卷积:(a)最大迭代次数50,(b)信噪比40,(c)质量阈值0.01,(d)迭代模式已优化。
      3. 将解卷积后的延时图像序列另存为TIFF z堆栈图像序列文件,以生成2D和3D延时视频。
    2. 2D和3D以及明场视频生成
      1. 对于2D视频生成,将解卷积的延时z堆栈导入Icy软件(步骤E1c)。选择聚焦最佳的单个z平面,调整亮度和对比度,并裁剪感兴趣的区域。将文件另存为.avi文件格式。
      2. 对于3D视频重建,将解卷积的z堆栈延时文件导入NIS元素,并对齐以校正X-Y漂移(步骤E1c)。使用NIS-Elements的“体积视图”和“电影制作器”模块生成3D视频。
      3. 对于2D明场视频生成,请将原始图像z-tack文件导入斐济(ImageJ)软件。选择聚焦最佳的单个z平面,调整亮度和对比度。将文件另存为.avi视频文件。
        注意:明场图像不需要进行反卷积。
      4. 要在2D荧光和明场视频上添加注释,请在Microsoft PowerPoint(MS Office 2016)上将.avi视频文件作为视频文件插入,调整幻灯片大小以与视频窗口尺寸匹配,然后使用“文本和形状”工具对视频进行注释。将PowerPoint文件导出为高分辨率.mp4电影文件。 (请参阅Akram et al。的补充电影,2019年)。

数据分析

  1. 量化细胞行为
    1. 为了量化PCLS产后肺泡生成过程中的细胞事件,例如细胞聚集,凹陷,分隔和细胞扩展,请仔细检查屏幕上已解卷积的明场延时视频文件(使用40倍物镜)。记录视频文件在单个视野中观察到每个事件的次数。检查来自不同PCLS和至少3个独立实验的至少4个不同领域的视频。
    2. 将数据表示为每种细胞行为的频率,作为每场对该行为观察的总数。 (有关结果的示例,请参阅《结果》部分,Akram等人的第4-7页,,2019年)

  2. 定量细胞迁移
    1. Icy中的跟踪管理器会从跟踪数据中生成一系列单元迁移信息,包括:净单元迁移,总位移,迁移速度(图4A)。
    2. 为了量化和比较不同出生后年龄组(例如,P3,P7,P14和成人)之间的上皮细胞迁移或不同的治疗条件,确定(a)净细胞迁移和(b)净迁移距离在指定的时间段内的单个单元格。净细胞迁移是计算得出的平均值,以表示细胞在X-Y轴(起始点A到端点B)之间迁移了多少线性距离。来自每个场的细胞总数的迁移距离的平均值表示为平均净细胞迁移。 (有关结果的示例,请参见Akram et al。中的图2 h,i,j,2019年)。
      注意:要进行迁移分析,请使用单元格跟踪记录中的“净位移”而不是“总位移”数据(图4A,黄色突出显示)。
    3. 肺切片中存在两组上皮细胞:一组细胞是无柄的,另一组细胞是高度运动的。为了区分这两组,并在不同的出生年龄组和治疗条件之间进行比较,请确定各个细胞的净移动距离。对它们进行排名并以迁移排名距离的细胞百分比或比例表示(请参见Akram et al。,2019年的图2 h-j和图6 k-n)。

菜谱

  1. HBSS / HEPES冰冷缓冲液
    1个HBSS
    1%HEPES 1 M
  2. 琼脂糖溶液
    1.5克低熔点琼脂糖粉
    100 ml HBSS / HEPES缓冲液
    将琼脂糖溶于微波的HBSS / HEPES缓冲液中。准备30毫升琼脂糖以处理1肺
  3. SF-DMEM
    DMEM基础培养基
    1%青霉素-链霉素
  4. 图像媒体
    具有HEPES基础培养基的无酚红DMEM
    1%青霉素-链霉素
    EpCAM-FITC抗体1:500
    PECAM抗体1:500或SiR-DNA 1:1,000
  5. MTT工作解决方案
    DMEM基础培养基
    10%MTT储备溶液(在DMSO中储备浓度为5 mg / ml)
    注意:储存溶液在-20°C的黑暗容器中保存时,可以稳定保存1年。
  6. 70%乙醇
    70毫升无水乙醇
    30毫升蒸馏水
  7. 70%甲醇
    70毫升甲醇
    30毫升PBS

致谢

该项目由Leverhulme Trust向CHD拨款(RPG-2015-226)资助。伦敦帝国学院的光学显微镜成像设备(FILM)由惠康基金会(Wellcome Trust)(拨款104931 / Z / 14 / Z)和BBSRC(拨款BB / L015129 / 1)的部分支持。该协议改编自Akram et al。,2019年发布的方法。

利益争夺

作者宣称他们没有利益冲突。

伦理

所有动物的维护和程序均按照1986年《动物(科学程序)法》的要求进行。动物工作得到伦敦帝国学院南肯辛顿和圣玛丽AWERB委员会的批准。

参考文献

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引用:Akram, K. M., Yates, L. L., Mongey, R., Rothery, S., Gaboriau, D. C. A., Sanderson, J., Hind, M., Griffiths, M. and Dean, C. H. (2019). Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices. Bio-protocol 9(20): e3403. DOI: 10.21769/BioProtoc.3403.
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