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Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting
采用流式细胞术分选术分离高纯度原代小鼠II型肺泡上皮细胞   

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参见作者原研究论文

本实验方案简略版
American Journal of Respiratory Cell and Molecular Biology
Jun 2016

Abstract

In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.

Background

The internal surfaces of lungs are lined by epithelial cells, the type of epithelial cell varying morphologically and functionally with the location within the lung. ATII cells are one of the two types of epithelial cells that line the alveolar walls and have been described to play critical roles in surfactant synthesis and secretion. They are also part of the first line of defense within the lung and are involved in initiating and modulating immune responses during pulmonary infection or allergy. They are also thought to act as progenitor cells in the distal lung with proliferative capacity and the ability to repair the epithelium after injury. Available methods of ATII isolation did not yield cell preps that were more than 80-85% pure, making them unsuitable for reliable analyses of mRNA and protein expression. The method described here is an improvement over prior methods and yields mouse primary ATII cell preps with the highest purity that can thus be reliably used for expression analyses. For further discussion on the method, we refer the reader to the original publication from where this protocol originates (Sinha et al., 2016).

Materials and Reagents

  1. C57BL/6 mice (Charles River Laboratories, strain code: 027)
  2. 2,2,2-tribromoethanol (Avertin) (Sigma-Aldrich, catalog number: T48402 )
  3. tert-Amyl alcohol (Sigma-Aldrich, catalog number: 240486 )
  4. 70% ethanol (Decon Labs, catalog number: V1401 )
  5. 1 ml slip-tip syringe (important to not use a Luer-LokTM tip syringe) (BD, catalog number: 309659 )
  6. 10 ml syringe (BD, Luer-LokTM, catalog number: 309604 )
  7. 25 G x 5/8 inch needle (BD, catalog number: 305122 )
  8. 20 G x 1.16 inch angiocatheter (BD, AngiocathTM, catalog number: 381134 )
  9. Surgical silk suture 3-0 (Henry Schein, catalog number: 100-7842 )
  10. 70 μm cell strainer (Corning, catalog number: 431751 )
  11. 40 μm cell strainer (Corning, catalog number: 352340 )
  12. 20 μm nylon mesh (Spectrum, catalog number: 146510 )
  13. Kimwipes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 06-666A )
  14. 5 ml polypropylene tubes (Corning, Falcon®, catalog number: 352063 )
  15. 15 ml tubes (Corning, catalog number: 430052 )
  16. 50 ml conical tube (Corning, catalog number: 430290 )
  17. Non TC-treated 10 cm Petri dish (Corning, catalog number: 430591 )
  18. D-PBS (without Ca2+ and Mg2+, no phenol) (Thermo Fisher Scientific, catalog number: 14190250 )
  19. 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  20. Dispase II (neutral protease, grade II) (Sigma-Aldrich, catalog number: 4942078001 )
  21. Low melting point agarose (Sigma-Aldrich, catalog number: A9419 )
  22. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  23. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  24. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  25. ACK lysis buffer (Lonza, catalog number: 10-548E )
  26. Biotinylated anti-mouse CD45, clone 30-F11, 0.5 mg/ml (Affymetrix, eBioscience, catalog number: 13-0451-85 )
  27. Biotinylated anti-mouse CD16/32, clone 2.4G2, 0.5 mg/ml (BD, PharmingenTM, catalog number: 553143 )
  28. Biotinylated anti-mouse CD31, clone MEC13.3, 0.5 mg/ml (Biolegend, catalog number: 102504 )
  29. Biotinylated anti-mouse TER119, clone TER119, 0.5 mg/ml (Affymetrix, eBioscience, catalog number: 13-5921-85 )
  30. Biotinylated anti-mouse Integrin β4, clone 346-11A, 0.5 mg/ml (Biolegend, catalog number: 12603 )
  31. Anti-mouse EpCAM-APC, clone G8.8, 0.2 mg/ml (Affymetrix, eBioscience, catalog number: 17-5791-82 )
  32. Dynabeads® MyOneTM streptavidin T1 magnetic beads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 65601 )
  33. Streptavidin-PE, 0.5 mg/ml (BD, PharmingenTM, catalog number: 554061 )
  34. Rabbit anti-pro-SP-C, polyclonal serum (EMD Millipore, catalog number: AB3786 )
  35. DNase I (Sigma-Aldrich, catalog number: D5025 )
  36. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140 )
  37. DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
  38. Avertin solution (100% stock working solution) (see Recipes)
  39. Avertin solution (2.5% stock working solution) (see Recipes)
  40. Dispase solution (see Recipes)
  41. 1% Low melting agarose (see Recipes)
  42. Complete DMEM (see Recipes)
  43. DNase I solution (see Recipes)
  44. Sort buffer (see Recipes)

Equipment

  1. Fine-tipped small scissors (Roboz surgical instrument company, catalog number: RS-5910 )
  2. Fine forceps (Roboz surgical instrument company, catalog number: RS-5211 )
  3. Horizontal platform orbital rocker (VWR, catalog number: 40000-300 )
  4. Tube rotator (Thermo Fisher Scientific, catalog number: 400110Q )
  5. Orbital incubator-shaker (VWR, catalog number: 10027-214 )
  6. Biosafety cabinet (VWR, catalog number: 89413-126 )
  7. Stratalinker (Stratagene, model: 1800 )
  8. DynaMagTM-2 magnetic separator (Thermo Fisher Scientific, catalog number: 12321D )
  9. FACS Aria cell sorter (BD, model: BD FACSARIA III )

Software

  1. FACS Diva (BD Biosciences)
  2. FlowJo (TreeStar)

Procedure

Note: Mice should be cared for and used in accordance with national and institutional policies. All protocols must be approved by the institutional animal committee. In our case, all procedures were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco.

  1. Mouse dissection
    1. Sacrifice mice using an approved inhaled or injectable anesthetic overdose procedure. We sacrificed mice by i.p. injection of 1 ml of 2.5% avertin per mouse.
      Note: Both CO2 asphyxiation and cervical dislocation are incompatible for the purposes of this protocol. Unlike anesthetic overdose, CO2 asphyxiation stops the heart from beating which then reduces perfusion quality later in the protocol. Cervical dislocation destroys structures in the neck region which are required intact for this protocol.
    2. Pin mouse down onto dissecting tray. With the help of a suture loop (Figure 1A) around the front incisors, immobilize the head (Figure 1B). Spray the ventral surface with 70% ethanol.
      Note: Immobilizing the head helps with tracheal cannulation and dispase infusion steps later.
    3. Cut open the fur, then cut open the peritoneum along the midline. Cut through the rib cage along the midline (Figure 1C) except the top part, which houses large blood vessels.
      Note: Take care to avoid nicking large blood vessels, the lungs or heart. The success of the following steps depends on the integrity of the above structures.
    4. Hold the rib cage with forceps and snip away the diaphragm from the place it attaches to the rib cage (Figure 1D). Then cut up on two sides of the midline to remove the sternum and expose the heart and lungs (Figure 1E).


      Figure 1. Mouse dissection steps. Immobilize the head of the mouse (A-B) and dissect the rib cage and diaphragm to expose lungs and heart (C-E).

  2. Cardiac perfusion
    Note: A video of cardiac perfusion has been provided for the user (Video 1).
    1. Snip the inferior vena cava (IVC) and aorta near the kidneys to exsanguinate the mouse.
      Note: Quickly proceed to perfusion as clots start to form as soon as the mouse starts bleeding, which drastically reduces the quality of perfusion.
    2. Take a D-PBS-filled 10 ml syringe attached to a 25 G x 5/8 inch needle and insert the needle into the right ventricle (Figure 2A), aiming in the direction of the pulmonary artery (Figure 2B). Gently push the plunger of the syringe to perfuse the lungs with 5 ml of D-PBS.
      Notes:
      1. You should see perfusate flowing out from the snip made in the IVC/aorta. With good perfusion, the lungs should turn white in color.
      2. Perfusion with plain D-PBS instead of D-PBS-EDTA is critical as EDTA inhibits dispase enzyme action downstream.
      3. You can hold the tip of the heart with forceps to prevent the needle from sliding out (Figure 2A).
    3. At the end of the perfusion, cut the heart away (Figure 2C).


      Figure 2. Cardiac perfusion of the lungs via right ventricle. Snip the IVC and insert needle into the right ventricle (A) pointing in the direction of the pulmonary artery (B). Lungs after perfusion with 5 ml DPBS (heart removed) (C).

      Video 1. Cardiac perfusion of mouse lungs via the right ventricle

  3. Tracheal cannulation
    1. Flip the dissection board such that the head of the mouse is closer to you.
      Note: This makes tracheal cannulation, dispase infusion and downstream manipulations much easier.
    2. Grab the two lobes of the salivary glands with fine forceps and gently pull them apart (Figures 3A and 3B).
      Note: The trachea lies just below the salivary glands.
    3. Expose the trachea by snipping the tissue covering it with fine-tipped small scissors.
    4. With the help of fine forceps, loop the trachea with surgical suture and make a loose knot (Figure 3C).
    5. With fine-tipped scissors, make a small nick on the ventral surface of the trachea.
      Note: The nick should be just big enough to pass the sheath of the 20 G angiocatheter, which serves well as a tracheal cannula (Figure 3D).
    6. Slide the sheath of the 20 G angiocatheter into the trachea and secure in place by tightening the suture knot around it (Figure 3E).
      Note: Do not slide the cannula too far down the trachea to avoid the cannula rupturing the airways at the point of bifurcation of the trachea into the two main extra-pulmonary bronchi or alternatively the cannula entering one of those bronchi, which will prevent all the lung lobes from being infused with dispase later on.


      Figure 3. Tracheal cannulation. Tease apart salivary glands (A-B) and expose the trachea. Loop the trachea with suture (C), insert cannula and secure in place (D-E).

  4. Dispase and agarose infusions
    1. Fill a slip-tip 1 ml syringe to maximal capacity with dispase (see Recipes).
    2. Attach the syringe to the tracheal cannula and slowly inject the full volume of dispase in the syringe. Leave the syringe in place for ~45 sec to allow the dispase to distribute throughout the lungs without allowing it to spill back out (Figure 4A).
      Note: You should see all the lung lobes inflate. If that is not the case, see tip under tracheal cannulation above.
    3. Fill another 1 ml slip-tip syringe with 0.6 ml of lukewarm (i.e., 42-45 °C) 1% low-melt agarose (see Recipes).
    4. Remove the syringe with dispase and quickly attach the agarose-containing syringe to the catheter.
      Note: Switching of the syringes should be done rapidly to minimize back flow and leakage of dispase. Having a slip-tip, instead of a Luer-Lok tip syringe greatly helps with speed at this step.
    5. Inject the agarose gently into the lungs, leave the syringe in place and cover the lungs with crushed ice for 2 min to allow for the agarose to solidify (Figure 4B).
      Rationale: The agarose pushes the enzyme further into the alveolar spaces, effectively plugs the airways and prevents club cells from digesting off the airways and ultimately contaminating the ATII epithelial preparation.
    6. Loosen the suture knot and slide the syringe and cannula out.


      Figure 4. Infusions of dispase followed by agarose. Inject dispase into lungs followed by agarose solution (A) and cover lungs with ice (B) for solidification of agarose.

  5. Dispase digestion
    1. Carefully dissect out the lungs from the thoracic cavity by grasping the trachea with forceps, then snipping down behind the lungs while gently pulling the lungs away. Avoid nicking the lungs.
    2. Rinse the lungs with D-PBS in a Petri dish till there is no more blood contaminating the outside surface of the lungs (Figure 5A). Dab away excess D-PBS with Kim wipes.
    3. Cut away individual lung lobes while excluding extra-pulmonary airways and other tissues (Figure 5B). Drop the lobes in a 50 ml conical tube containing 0.5 ml of dispase (Figure 5C).
      Note: Do not nick or chop the lung lobes. The lobes need to be kept intact for selective and efficient release of ATII cells. Chopping up will cause release of contaminating cell types from the airways.
    4. Incubate for 45 min at room temperature (RT) on a rocker at 150 rpm.
      Notes:
      1. Digestion should not be done at 37 °C as increased dispase activity at 37 °C release many more types and numbers of contaminating stromal cells from the lungs which is undesirable. At RT, the digestion preferentially releases ATII cells.
      2. Rocking helps with digestion but the exact speed of rocking is not critical. In winter months, if the laboratory temperature drops significantly lower than 25 °C, a forced-air incubator-shaker set to 25 °C can be used as an alternative as low temperatures lead to poor lung tissue digestion.


        Figure 5. Dispase digestion. Rinse lung lobes in DPBS (A), snip away individual lobes leaving out airways (B) and transfer to 50 ml conical with more dispase for digestion (C).

  6. Preparation of single cell prep
    1. Decant digested lungs from the 50 ml conical into a 10 cm Petri dish (Figure 6A). Add 7 ml of complete DMEM and 10 μl of DNase (see Recipes).
      Note: Without DNase, the cells will be very sticky and clump together, which eventually lowers cell yield at the end.
    2. Gently tease the lung parenchyma away from the large airways using sharp tweezers. Avoid teasing near large airways (Figures 6B and 6C).


      Figure 6. Preparation of crude lung single cell suspension. After 45 min digestion (A), gently tease lung parenchyma with fine forceps (B-D) to release more cells into suspension.

    3. Rock the Petri dish in a horizontal plane at 60 rpm for another 10 min at RT to allow more cell release.
      Note: Rocking helps with digestion but the exact speed of rocking is not critical.
    4. Use two fine forceps to keep the digested lung tissue in a compact mass and repeatedly move one pair of forceps down the tissue mass to release more cells (Figure 6D).
    5. Discard the airways and serially strain the lung crude single cell prep through a 70 μm, 40 μm and 20 μm* strainers.
      Notes:
      1. At the end of each straining step, rinse the Petri dish or 50 ml conical and strainer with 2 ml complete DMEM and pool with cell suspension to maximize cell recovery.
      2. *On the assembly of 20 μm strainers: To prepare your own 20 μm strainers (not available commercially), cut out the base from a 50 ml conical and also cut out a hole in the cap (Figure 7A) using a sharp blade and caution. Cut a square from the 20 μm nylon mesh sheet and cover the top of the cut 50 ml conical (Figure 7B). Screw the nylon mesh in place with the help of the cut cap (Figures 7C and 7D). To strain cells, place strainer cap side down on top of a fresh 50 ml conical. If the isolated cells will be cultured ex vivo, the 20 μm strainer can be sterilized prior to use with 120 Joules/meter2 dose of UV light delivered using a Stratalinker model 1800. Please note that the nylon mesh is not autoclavable.


        Figure 7. Assembly of 20 μm strainer. Using a cut 50 ml conical (A) and a square piece of 20 μm nylon mesh (B), assemble the strainer (C-D).

    6. Count cells. At this step, the cell yield is ~25-35 million cells per mouse lung.
    7. Spin cells down at 300 x g, 10 min, 4 °C.
      Optional: If the pellet shows presence of RBCs, gently resuspend pellet in 5 μl DNase and 2 ml ACK lysis buffer. Place on ice for 2 min. Make up volume to 10 ml with complete DMEM and spin cells down at 300 x g, 10 min, 4 °C.
    8. Resuspend cell pellet in 5 μl DNase and 500 μl complete DMEM.

  7. Magnetic enrichment of ATII cells
    1. Stain with biotinylated antibodies against lineage (Lin) markers – add 5 μl of each of the following biotinylated antibodies to single cells (from one lung) suspended in 500 μl of complete DMEM and 5 μl of DNase: anti-CD45, anti-CD16/32, anti-CD31, anti-Ter119, anti-integrin β4.
      Rationale: The above antibody cocktail will mark hematopoietic cells (CD45 positive), alveolar macrophages (CD45 positive and CD16/32 positive), endothelial cells (CD31 positive), erythroid cells (Ter119 positive), club cells (integrin β4 positive) and distal lung progenitor cells (integrin β4 positive) for depletion.
      Note: The above antibody amounts are optimal when isolating ATII cells from naïve mice lungs. When isolating ATII cells from inflamed lungs with increased numbers of inflammatory cell infiltrates, the amounts of antibodies against lineage markers can be increased at this magnetic depletion step. The user should optimize this based on their mouse model and needs.
    2. Incubate for 45-60 min on ice with intermittent mixing by gentle flicking of the cells.
    3. While the cells are incubating with the antibodies, prepare the magnetic beads for use (2.5 µl SA MyOne T1 beads/1 x 106 cells). Wash the beads with 1 ml D-PBS twice to remove azide. Collect beads in the DynaMag-2 magnetic separator.
    4. At the end of the antibody incubation, wash the cells once in 10 ml complete DMEM (spin at 300 x g, 10 min, 4 °C) and resuspend in 500 μl complete DMEM and 5 μl DNase. Transfer cells to 1.5 ml tube with the appropriate number of washed beads. Incubate in the cold room for 30 min on a tube rotator to prevent the beads from settling to the bottom.
    5. Magnetic separation – Place the 1.5 ml tube with bead-bound cells in the magnetic separator for 2 min. Lineage positive cells will get stuck to the tube wall. Gently pipette out unattached cells and place into a fresh 1.5 ml tube. Place the fresh tube in the magnetic and repeat magnetic depletion for a total of 3 times.

  8. Cell sorting
    1. Transfer magnetically enriched ATII cells to a 5 ml FACS tube.
    2. Re-stain cells for lineage markers using biotinylated antibodies exactly as before.
    3. Wash cells as before with complete DMEM and resuspend in 500 μl complete DMEM and 5 μl of DNase.
    4. Add 1 μl of streptavidin-PE (to place all the lineage marker positive cells in the PE channel) and 5 μl of EpCAM-APC (to stain all epithelial cells). Incubate for 45-60 min on ice in the dark with intermittent mixing by gentle flicking of the cells.
    5. Wash cells with 10 ml sort buffer (see Recipes) once and resuspend at 5 x 106 cells/ml in sort buffer with DAPI (0.5 μg/ml) for FACS sorting. Strain cells through a 40 μm strainer to avoid any cell clumps that might clog the cell sorter.
    6. Set up the FACS Aria cell sorter for sorting using a 100 μm nozzle. Set up compensation using single color controls.
      Note: Please note that covering these aspects in detail is beyond the scope of this protocol. A new user can perhaps get help of a flow cytometry core operator for detailed instructions on cell sorting depending on the sorter available to the user.
    7. Sort FSChi SSChi, singlet, DAPI- live, Lin- EpCAM+ cells (Figure 8), which are ATII cells, into 15 ml tubes containing 5 ml of ice-cold complete DMEM. Typically, this protocol yields 2-3 x 106 highly pure (98-99%) and highly viable (97-98%) ATII cells per lung from a naïve mouse.


      Figure 8. FACS sorting strategy for isolating highly pure murine ATII cells. Shown are representative flow cytometric plots of lung cells collected before and after magnetic enrichment, and after sorting. Displayed on the y-axis is staining for Lin markers (CD45, CD16/32, CD31, Ter119 and integrin β4) while the x-axis shows EpCAM staining. Events displayed on the plots are DAPI negative single cells. A gate around Lin- EpCAM+ cells, as shown above, is used to sort the cells.

Data analysis

  1. At the end of cell sorting, rerun sorted cells through the FACS Aria to check for purity. We have routinely observed that the sorted cells are 98-99% Lin- EpCAM+ and 97-98% viable (Figure 8).
  2. Optional: Purity can also be assessed by the expression of pro-SP-C, which is a specific marker for ATII cells. About 30,000 sorted cells can be cytospun and then stained for pro-SP-C to check for purity (Figure 9). Alternatively, ~200,000-500,000 cells can be saved for intracellular staining for pro-SP-C using standard intracellular flow cytometry methods. The previous method allows visualization of cell morphology – pro-SP-C staining is found in the cytoplasm and is granular in appearance (Figure 9, inset). The latter method yields superior quantitation of ATII prep purity. We routinely found that our sorted cells were 98-99% pro-SP-C+. For more details, please refer to the original publication (Sinha et al., 2016).


    Figure 9. Purity of sorted ATII cells. Cells from (A) crude lung digest and (B) ATII cells after FACS sorting were spun onto slides and stained for pro-SP-C, a specific ATII marker, to assess purity.

  3. Isolated ATII cells can be used for different purposes. For RT-PCR analysis, sorted cells can be pelleted and subsequently lysed in lysis buffer for RNA isolation (as per your choice of RNA isolation method). For protein expression analysis by Western blotting, wash the cells once with 10 ml of D-PBS to remove serum proteins – this wash step is critical to avoid serum proteins from interfering with your Western blotting. Spin down cells and resuspend pellet in lysis buffer supplemented with protease inhibitor cocktail (as per your choice of protein extraction method). Alternatively, cell pellets can be snap frozen and stored in liquid nitrogen till the RNA or protein extraction step. For ex vivo culture, these cells can be grown on matrigel or fibronectin-coated surfaces or in other ways depending on your experimental question.

Notes

  1. If the isolated cells need to be cultured, all steps must be carried out in a biosafety cabinet and usual sterile procedures should be followed. Dissection instruments must be autoclaved.
  2. It is recommended that a new user unfamiliar with the techniques practice and gain some technical experience with each step prior to attempting the full protocol for isolation. This will increase the chances of a successful isolation.
  3. The protocol is fairly long. Depending on the number of mice to be sacrificed and the desired yield of ATII cells, the full procedure could take 7-8 h.

Recipes

  1. 100% avertin stock solution
    10 g 2,2,2-tribromoethanol
    10 ml tert-amyl alcohol
    Dissolve by heating and stirring
    Solution is light sensitive; keep covered in foil and store at 4 °C
  2. 2.5% avertin working solution
    Prepare a 2.5% avertin working solution by diluting the 100% stock 1:40 in DPBS or isotonic saline
    e.g., 1 ml of 100% avertin stock plus 39 ml DPBS. Filter working solution with 0.22 µm strainer
    Solution is light sensitive; keep covered in foil and store at 4 °C
    Note: Label container with the date of dilution and use within 30 days of that date.
  3. Dispase solution
    50 U/ml in D-PBS
    Reconstitute an entire 1 g vial at once
    Store aliquots at -20 °C
    Note: Avoid multiple freeze-thaw cycles; thaw on ice prior to use.
  4. Low melting agarose (1%)
    50 mg in 5 ml water
    Using a boiling water bath, boil agarose till clear
    Ready to use when lukewarm to touch (about 42-45 °C)
    Prepare fresh
  5. Complete DMEM (DMEM with 10% FBS, Pen-Strep, 10 mM HEPES)
    500 ml DMEM
    50 ml heat-inactivated fetal bovine serum
    5 ml Pen-Strep (10,000 U/ml)
    5 ml of 1 M HEPES
    Prepare in a biosafety cabinet under sterile conditions; store at 4 °C; maintain sterility
  6. DNase I solution
    5,000 Kunitz U/ml in D-PBS
    Store aliquots at -20 °C
    Note: Avoid multiple freeze-thaw cycles; thaw on ice prior to use.
  7. Sort buffer (D-PBS with 2% FBS, 1 mM EDTA)
    500 ml D-PBS (without Ca2+ and Mg2+)
    10 ml heat-inactivated fetal bovine serum
    1 ml of 0.5 M EDTA
    Prepare in a biosafety cabinet under sterile conditions; store at 4 °C; maintain sterility

Acknowledgments

This work was supported by the US National Institutes of Health (AI65495 and AI68150 to C.A.L.). Thanks to Pooja Mehta and James Mueller for help with pictures and video. This protocol was adapted from Sinha et al. (2016).

References

  1. Sinha, M. and Lowell, C. A. (2016). Immune defense protein expression in highly purified mouse lung epithelial cells. Am J Respir Cell Mol Biol 54(6): 802-813.

简介

在这个协议,我们描述从初始小鼠的肺分离高纯度原发性肺泡上皮细胞类型(ATII)细胞的方法。该方法结合对多种谱系标志物的阴性选择以及对于Ep上皮细胞标记物EpCAM的阳性选择。该方法每小鼠肺产生2-3×10 6个ATII细胞。细胞制品是高度纯的和可行的,并且可以用于基因组或蛋白质组分析或培养离体以了解他们在各种生物过程中的作用。

[背景] 肺的内表面由上皮细胞排列,上皮细胞的类型在形态上和功能上随着肺内的位置而变化。 ATII细胞是两种类型的上皮细胞中的一种,其排列在肺泡壁上并且已经被描述为在表面活性剂合成和分泌中起关键作用。它们也是肺内第一道防线的一部分,并且涉及在肺部感染或过敏期间引发和调节免疫应答。它们还被认为在远端肺中充当具有增殖能力和损伤后修复上皮的能力的祖细胞。 ATII分离的可用方法不产生超过80-85%纯度的细胞制品,使得它们不适合于mRNA和蛋白质表达的可靠分析。本文所述的方法是对现有方法的改进,并产生具有最高纯度的小鼠原代ATII细胞制备物,因此可以可靠地用于表达分析。对于该方法的进一步讨论,我们将读者指向该协议起源的原始出版物(Sinha等人,2016)。

材料和试剂

  1. C57BL/6小鼠(Charles River Laboratories,菌株代码:027)
  2. 2,2,2-三溴乙醇(Avertin)(Sigma-Aldrich,目录号:T48402)
  3. 叔戊醇(Sigma-Aldrich,目录号:240486)
  4. 70%乙醇(Decon Labs,目录号:V1401)
  5. 1ml滑动注射器(不使用Luer-Lok TM 尖端注射器)(BD,目录号:309659)
  6. 10ml注射器(BD,Luer-Lok TM ,目录号:309604)
  7. 25G×5/8英寸针(BD,目录号:305122)
  8. 20 G×1.16英寸血管导管(BD,Angiocath TM ,目录号:381134)
  9. 外科缝线3-0(Henry Schein,目录号:100-7842)
  10. 70μm细胞过滤器(Corning,目录号:431751)
  11. 40μm细胞过滤器(Corning,目录号:352340)
  12. 20μm的尼龙网(Spectrum,目录号:146510)
  13. Kimwipes(Thermo Fisher Scientific,Fisher Scientific,目录号:06-666A)
  14. 5ml聚丙烯管(Corning,Falcon ,目录号:352063)
  15. 15ml管(Corning,目录号:430052)
  16. 50ml锥形管(Corning,目录号:430290)
  17. 非TC处理的10cm培养皿(Corning,目录号:430591)
  18. D-PBS(不含Ca 2+和Mg 2+ 2+,不含苯酚)(Thermo Fisher Scientific,目录号:14190250)
  19. 0.5M EDTA,pH 8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  20. Dispase II(中性蛋白酶,II级)(Sigma-Aldrich,目录号:4942078001)
  21. 低熔点琼脂糖(Sigma-Aldrich,目录号:A9419)
  22. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
  23. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  24. 1 M HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  25. ACK裂解缓冲液(Lonza,目录号:10-548E)
  26. 生物素化的抗小鼠CD45,克隆30-F11,0.5mg/ml(Affymetrix,eBioscience,目录号:13-0451-85)
  27. 生物素化的抗小鼠CD16/32,克隆2.4G2,0.5mg/ml(BD,Pharmingen ,目录号:553143)
  28. 生物素化的抗小鼠CD31,克隆MEC13.3,0.5mg/ml(Biolegend,目录号:102504)
  29. 生物素化的抗小鼠TER119,克隆TER119,0.5mg/ml(Affymetrix,eBioscience,目录号:13-5921-85)
  30. 生物素化的抗小鼠整合素β4,克隆346-11A,0.5mg/ml(Biolegend,目录号:12603)
  31. 抗小鼠EpCAM-APC,克隆G8.8,0.2mg/ml(Affymetrix,eBioscience,目录号:17-5791-82)
  32. 链霉抗生物素蛋白T 1磁珠(Thermo Fisher Scientific,Invitrogen TM ,目录号:65601)的微珠中加入
  33. 链霉抗生物素-PE,0.5mg/ml(BD,Pharmingen,目录号:554061)
  34. 兔抗前SP-C,多克隆血清(EMD Millipore,目录号:AB3786)
  35. DNase I(Sigma-Aldrich,目录号:D5025)
  36. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:26140)
  37. DAPI(Thermo Fisher Scientific,Molecular Probes ,目录号:D1306)
  38. Avertin溶液(100%原液工作溶液)(参见配方)
  39. Avertin溶液(2.5%储存工作溶液)(参见配方)
  40. Dispase解决方案(参见配方)
  41. 1%低熔点琼脂糖(见配方)
  42. 完成DMEM(参见配方)
  43. DNase I解决方案(参见配方)
  44. 排序缓冲区(请参阅配方)

设备

  1. 细尖小剪刀(Roboz外科器械公司,目录号:RS-5910)
  2. 精细镊子(Roboz外科器械公司,目录号:RS-5211)
  3. 水平平台轨道摇臂(VWR,目录号:40000-300)
  4. 管旋转器(Thermo Fisher Scientific,目录号:400110Q)
  5. 轨道式摇床(VWR,目录号:10027-214)
  6. 生物安全柜(VWR,目录号:89413-126)
  7. Stratalinker(Stratagene,型号:1800)
  8. DynaMag TM -2磁性分离器(Thermo Fisher Scientific,目录号:12321D)
  9. FACS Aria细胞分选仪(BD,型号:BD FACSARIA III)

软件

  1. FACS Diva(BD Biosciences)
  2. FlowJo(TreeStar)

程序

注意:小鼠应根据国家和机构政策进行照顾和使用。所有方案必须经机构动物委员会批准。在我们的例子中,所有程序都由加利福尼亚州旧金山大学的机构动物护理和使用委员会批准。

  1. 鼠标解剖
    1. 牺牲小鼠使用批准的吸入或可注射麻醉过量程序。我们通过腹腔注射处死小鼠。注射1ml的2.5%avertin /小鼠 注意:为了本协议的目的,CO 2窒息和颈椎脱臼是不相容的。与麻醉剂过量不同,CO 2窒息使心脏停止跳动,然后在方案中稍后降低灌注质量。颈椎脱臼会破坏颈部区域的结构,这是本协议要求的完整。
    2. 将鼠标按下到解剖托盘。借助于在前切牙周围的缝线环(图1A),固定头部(图1B)。用70%乙醇喷洒腹侧表面 注意:固定头部有助于气管插管和以后分配输注步骤。
    3. 切开毛皮,然后沿中线切开腹膜。沿着中线切开肋骨架(图1C),除了容纳大血管的顶部。
      注意:注意避免切割大血管,肺或心脏。以下步骤的成功取决于上述结构的完整性。
    4. 用钳子抓住肋骨架,将膜片从其连接到肋骨的位置剪断(图1D)。然后在中线的两侧切开以去除胸骨,暴露心脏和肺(图1E)。


      图1.鼠标解剖步骤。固定鼠标的头部(A-B),并解剖肋骨和隔膜以暴露肺和心脏(C-E)。

  2. 心脏灌注
    注意:已为用户提供了心脏灌注视频(视频1)。
    1. 切下下腔静脉(IVC)和肾脏附近的主动脉放血小鼠。
      注意:快速进行灌注,因为一旦鼠标开始出血,凝块开始形成,这大大降低了灌注的质量。
    2. 取连接到25 G x 5/8英寸针头的D-PBS填充的10ml注射器,并将针插入右心室(图2A),瞄准肺动脉的方向(图2B)。轻轻推动注射器的柱塞,用5ml D-PBS灌注肺部 注意:
      1. 你应该看到灌注液从IVC /主动脉制造的剪刀流出。有良好的灌注,肺部应该变成白色。
      2. 用纯D-PBS而不是D-PBS-EDTA灌注是至关重要的,因为EDTA抑制下游的分泌酶作用。
      3. 您可以用镊子夹住心尖,以防止针头滑出(图2A)。
    3. 在灌注结束时,切开心脏(图2C)。


      图2.通过右心室的心脏灌注肺。切断IVC,将针插入右心室(A),指向肺动脉方向(B)。用5ml DPBS(去除心脏)(C)灌注后的肺。

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  3. 气管插管
    1. 翻转解剖板,使鼠标的头部更接近你。
      注意:这使气管插管,分散输液和下游操作更容易。
    2. 用细镊子抓住唾液腺的两个瓣,轻轻地将它们拉开(图3A和3B)。
      注意:气管位于唾液腺下方。
    3. 通过用细尖的小剪刀剪掉覆盖它的组织暴露气管。
    4. 在细镊子的帮助下,用手术缝合气管,形成松结(图3C)。
    5. 用细尖的剪刀,在气管的腹侧表面做一个小刻痕。
      注意:切口应该足够大,以通过20 G血管导管的鞘,这可以作为气管插管(图3D)。
    6. 将20 G血管导管的护套滑入气管,并通过收紧其周围的缝线固定到位(图3E)。
      注意:不要将插管沿气管向下滑动太远,以避免插管在气管分叉进入两个主要的肺外支气管的点处使气道破裂,或者插入进入其中一支气管的插管防止以后所有的肺叶注入分泌。


      图3.气管插管。嘲笑唾液腺(A-B),暴露气管。用缝合线环绕气管(C),插入套管并固定到位(D-E)
  4. 分散酶和琼脂糖输注
    1. 填充一个滑头1毫升注射器的最大容量分散(见配方)。
    2. 将注射器连接到气管套管,并缓慢注射在注射器中的全部体积的分散酶。离开注射器到位约45秒,以允许分散在整个肺部分布,而不允许其溢出(图4A)。
      注意:你应该看到所有的肺叶膨胀。如果不是这种情况,请参阅上面的气管插管下的尖端。
    3. 在另一个1ml的滑动注射器中加入0.6ml微温(即,42-45℃)1%低熔点琼脂糖(参见Recipes)。
    4. 用分散液取出注射器,并迅速将含琼脂糖的注射器连接到导管 注意:应尽快更换注射器,以尽量减少回流和分流的泄漏。有一个滑头,而不是一个鲁尔洛克尖注射器大大有助于在这一步的速度。
    5. 将琼脂糖轻轻注入肺中,将注射器留在原位,用碎冰覆盖肺部2分钟以使琼脂糖固化(图4B)。
      原理:琼脂糖将酶进一步推入肺泡空间,有效地堵塞气道,并防止俱乐部细胞消化气道,并最终污染ATII上皮制剂。
    6. 松开缝线结,将注射器和套管滑出。


      图4.注射分散酶,随后是琼脂糖。注射分散到肺中,随后是琼脂糖溶液(A),并用冰(B)覆盖肺以固化琼脂糖。

  5. 分散酶消化
    1. 小心地从胸腔解剖出肺,用镊子抓住气管,然后在肺后面轻轻地拉出肺。避免切口肺。
    2. 在培养皿中用D-PBS冲洗肺,直到没有更多的血液污染肺的外表面(图5A)。用Kim擦拭巾擦掉多余的D-PBS。
    3. 切除单个肺叶,同时排除肺外气道和其他组织(图5B)。将叶片放入含有0.5ml分散酶的50 ml锥形管中(图5C) 注意:不要切口或切除肺叶。为了选择性和有效释放ATII细胞,需要保持叶片完整。切断将导致污染细胞类型从气道中释放。
    4. 在室温(RT)下在摇床上以150rpm孵育45分钟 注意:
      1. 消化不应在37℃进行,因为在37℃下增加的分散酶活性从肺释放更多类型和数量的污染性基质细胞,这是不期望的。在RT,消化优先释放ATII细胞。
      2. 摇摆有助于消化,但摇摆的确切速度不是关键。在冬季,如果实验室温度显着低于25℃,可以使用设定为25℃的强制空气培养箱 - 摇动器作为替代,因为低温导致差的肺组织消化。
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        图5.分散酶消化。在DPBS(A)中冲洗肺叶,剪掉离开气道的单个肺叶(B),转移至50ml锥形,用更多分散酶消化(C) >
  6. 单细胞制备物的制备
    1. 将消化的肺从50ml圆锥体吸入10cm培养皿(图6A)。加入7毫升完全DMEM和10微升DNase(见配方)。
      注意:如果没有DNase,细胞会非常粘并结块在一起,最终会降低细胞产量。
    2. 使用锋利的镊子轻轻地从大气道远离肺实质。避免在大型航空公司附近挑逗(图6B和6C)。


      图6.粗制肺单细胞悬浮液的制备 45分钟消化(A)后,用细镊子(B-D)轻轻挑取肺实质,释放更多的细胞悬浮。
    3. 将培养皿在水平平面中以60rpm摇动10分钟,以允许更多的细胞释放 注意:摇摆有助于消化,但摇摆的确切速度并不重要。
    4. 使用两个细镊子保持消化的肺组织在一个紧凑的质量,并重复移动一对镊子沿组织块释放更多的细胞(图6D)。
    5. 丢弃气道,并通过70μm,40μm和20μm*过滤器连续应用肺粗制单细胞制剂。
      注意:
      1. 在每个应变步骤结束时,用2ml完全DMEM冲洗陪替氏培养皿或50ml锥形和过滤器,并与细胞悬浮液一起使细胞回收最大化。
      2. 在20微米过滤器的组装上:为了制备自己的20微米过滤器(不可商购),从50ml圆锥体中切出基底,并且还使用尖锐的切口在帽中切出一个孔(图7A)刀片和警告。从20μm的尼龙网片切割正方形,并覆盖切割的50ml锥形的顶部(图7B)。借助于切割帽将尼龙网拧紧在适当位置(图7C和7D)。为了应变细胞,将过滤器帽侧向下放置在新鲜的50ml圆锥形的顶部。如果分离的细胞将在离体培养下进行培养,则可以使用120焦耳/米2剂量的使用Stratalinker 1800型递送的UV光来灭菌20μm过滤器。请注意,尼龙网不是可高压灭菌的。

        图7.装配20μm过滤器使用切割的50ml圆锥形(A)和20μm的尼龙网格(B)的方形块,组装过滤器(CD) br />
    6. 计数单元格。在此步骤中,细胞产量为每只小鼠肺约25-35百万个细胞
    7. 旋转细胞以300×g /分钟,10分钟,4℃ 可选:如果沉淀显示存在RBC,轻轻地将沉淀重悬在5μlDNA酶和2ml ACK裂解缓冲液中。放在冰上2分钟。使用完全DMEM将体积补足至10ml,并以300×g,10分钟,4℃下降。
    8. 重悬细胞沉淀在5μlDNA酶和500μl完全DMEM
  7. ATII细胞的磁性富集
    1. 用生物素化的针对谱系(Lin)标记的抗体染色 - 向悬浮在500μl完全DMEM和5μlDNA酶:抗CD45,抗CD16 /抗体的单细胞(来自一个肺)中加入5μl的以下生物素化抗体, 32,抗CD31,抗Ter119,抗整联蛋白β4 上述抗体混合物将标记造血细胞(CD45阳性),肺泡巨噬细胞(CD45阳性和CD16/32阳性),内皮细胞(CD31阳性),红细胞(Ter119阳性),淋巴细胞(整联蛋白β4阳性) )和远端肺祖细胞(整合素β4阳性)。
      注意:当从初始小鼠肺分离ATII细胞时,上述抗体量是最佳的。当用炎症细胞浸润数量增加从发炎的肺分离ATII细胞时,可以在该磁耗竭步骤中增加针对谱系标志物的抗体的量。用户应根据他们的鼠标模型和需要进行优化。
    2. 在冰上孵育45-60分钟,通过轻轻轻拂细胞,间歇混合
    3. 当细胞与抗体孵育时,制备使用的磁珠(2.5μlSA MyOne T1珠/1×10 6个细胞)。用1ml D-PBS洗涤珠两次以除去叠氮化物。在DynaMag-2磁选机中收集磁珠。
    4. 在抗体孵育结束时,在10ml完全DMEM(在300×g,10分钟,4℃下旋转)中洗涤细胞一次,并重悬于500μl完全DMEM和5μlDNA酶中。转移细胞到1.5毫升管与适当数量的洗涤珠。在冷室中在管式旋转器上孵育30分钟,以防珠子沉降到底部
    5. 磁分离 - 将带有珠结合细胞的1.5ml管置于磁分选器中2分钟。谱系阳性细胞将粘附到管壁。轻轻吸出未附着的细胞,并放入一个新鲜的1.5毫升管。将新鲜试管放在磁性和重复磁性消耗总共3次。

  8. 单元格排序
    1. 转移磁性富集的ATII细胞到5ml FACS管
    2. 使用生物素化的抗体与以前一样重新染色谱系标记的细胞。
    3. 如前所述用完全DMEM洗涤细胞,并重悬于500μl完全DMEM和5μlDNA酶中
    4. 加入1微升链霉亲和素PE(以放置所有谱系标记阳性细胞在PE通道)和5微升的EpCAM-APC(染色所有上皮细胞)。在冰上在黑暗中孵育45-60分钟,通过轻轻轻拂细胞而间歇混合。
    5. 用10ml分选缓冲液(参见Recipes)洗涤细胞一次,并以5×10 6个细胞/ml重悬在具有DAPI(0.5μg/ml)的分选缓冲液中用于FACS分选。应变细胞通过一个40微米过滤器,以避免任何细胞块,可能会阻塞细胞分选机
    6. 设置使用100μm喷嘴进行分选的FACS Aria细胞分选仪。使用单色控制设置补偿。
      注意:请注意,详细讨论这些方面超出了本协议的范围。新用户可能得到流式细胞术核心操作员的帮助,以根据用户可用的分拣机获得细胞分选的详细说明。
    7. 排序FSC hi SSC hi ,singlet,DAPI - live,Lin - EpCAM + 细胞(图8)(其为ATII细胞)转移到含有5ml冰冷的完全DMEM的15ml管中。通常,该方案从幼稚的小鼠每肺产生2-3×10 6个高纯度(98-99%)和高活力(97-98%)ATII细胞。


      图8.分离高纯度鼠ATI1细胞的FACS分选策略。显示了在磁性富集之前和之后收集的肺细胞的代表性流式细胞计数图。在y轴上显示Lin标记(CD45,CD16/32,CD31,Ter119和整联蛋白β4)的染色,而x轴显示EpCAM染色。在图上显示的事件是DAPI阴性单细胞。如上所示,围绕Lin - EpCAM + 单元格的栅极用于对单元格进行排序。

数据分析

  1. 在细胞分选结束时,通过FACS Aria重新分选分选的细胞以检查纯度。我们常规地观察到分选的细胞是98-99%Lin - EpCAM + 和97-98%活的(图8)。
  2. 可选:纯度也可以通过pro-SP-C的表达来评估,SP-C是ATII细胞的特异性标记。可以将约30,000个分选的细胞进行细胞离心,然后对前-SP-C染色以检查纯度(图9)。或者,可使用标准细胞内流式细胞术方法保存约200,000-500,000个细胞用于原-SP-C的细胞内染色。先前的方法允许可视化的细胞形态 - 前SP-C染色发现在细胞质中,并且在外观上是颗粒状的(图9,插图)。后一种方法产生更高的ATII制备纯度的定量。我们常规发现我们的分选细胞为98-99%pro-SP-C + 。有关详细信息,请参阅原始出版物(Sinha等人。,2016)。


    图9.分选的ATII细胞的纯度。将FACS分选后来自(A)粗制肺消化物和(B)ATII细胞的细胞旋转到载玻片上,并对前SP-C,特异性ATII标记,以评估纯度
  3. 分离的ATII细胞可用于不同的目的。对于RT-PCR分析,可以将分选的细胞沉淀并随后在裂解缓冲液中裂解以用于RNA分离(根据您选择的RNA分离方法)。对于通过Western印迹的蛋白质表达分析,用10ml D-PBS洗涤细胞一次以除去血清蛋白 - 该洗涤步骤对于避免血清蛋白干扰蛋白质印迹是至关重要的。旋转细胞和重悬浮在补充了蛋白酶抑制剂鸡尾酒(根据您选择的蛋白质提取方法)的裂解缓冲液。或者,可以将细胞沉淀快速冷冻并储存在液氮中,直到RNA或蛋白质提取步骤。对于离体培养,这些细胞可以在基质胶或纤连蛋白包被的表面上生长或以其它方式生长,这取决于实验问题。

笔记

  1. 如果需要培养分离的细胞,所有步骤必须在生物安全柜中进行,并且应遵循通常的无菌程序。解剖仪器必须高压灭菌。
  2. 建议一个不熟悉技术实践的新用户在尝试完整的协议隔离之前获得每个步骤的一些技术经验。这将增加成功隔离的机会。
  3. 协议相当长。根据待处死的小鼠数目和所需的ATII细胞产量,完整程序可能需要7-8小时。

食谱

  1. 100%avertin储备液
    10g 2,2,2-三溴乙醇 10ml叔戊醇 通过加热和搅拌溶解
    解决方案是光敏感;保持覆盖箔并存储在4°C
  2. 2.5%avertin工作溶液
    通过在DPBS或等渗盐水中稀释100%储备液1:40制备2.5%的avertin工作溶液。
    例如,1ml 100%avertin原液加上39ml DPBS。用0.22μm过滤器过滤工作溶液
    解决方案是光敏感;保持覆盖箔并存储在4°C
    注意:标签容器的稀释日期应在该日期后30天内使用。
  3. 分散溶液
    在D-PBS中为50U/ml 一次重新制成一个1克的小瓶
    将等分试样储存在-20°C
    注意:避免多次冻融循环;在使用前在冰上解冻。
  4. 低熔点琼脂糖(1%)
    50毫克在5毫升水中
    使用沸水浴,煮沸琼脂糖至澄清
    准备使用时温和触摸(约42-45°C)
    准备新鲜的
  5. 完全DMEM(含有10%FBS,Pen-Strep,10mM HEPES的DMEM)
    500 ml DMEM
    50ml热灭活的胎牛血清 5ml Pen-Strep(10,000U/ml) 5ml 1 H HEPES
    在无菌条件下在生物安全柜中进行准备;储存在4°C;保持无菌
  6. DNase I溶液
    5,000 Kunitz U/ml在D-PBS中
    将等分试样储存在-20°C
    注意:避免多次冻融循环;在使用前在冰上解冻。
  7. 分选缓冲液(含有2%FBS,1mM EDTA的D-PBS)
    500ml D-PBS(不含Ca 2+和Mg 2+))。 10ml热灭活的胎牛血清 1ml 0.5M EDTA
    在无菌条件下在生物安全柜中进行准备;储存在4°C;保持无菌

致谢

这项工作得到美国国立卫生研究院(AI65495和AI68150至C.A.L.)的支持。感谢Pooja Mehta和James Mueller对图片和视频的帮助。此协议改编自Sinha等人。 (2016年)。

参考文献

  1. Sinha,M.和Lowell,CA(2016)。  在高度纯化的小鼠肺上皮细胞中的免疫防御蛋白表达。 Am J Respir Cell Mol Biol 54(6):802-813。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sinha, M. and Lowell, C. A. (2016). Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting. Bio-protocol 6(22): e2013. DOI: 10.21769/BioProtoc.2013.
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