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Dec 2017
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Isolation and Quantification of Mouse γδT-cells in vitro and in vivo
体外和体内小鼠γδT细胞的分离和定量   

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Abstract

The skin plays an important role in protecting the body from pathogens and chemicals in the external environment. Upon injury, a healing program is rapidly initiated and involves extensive intercellular communication to restore tissue homeostasis. The deregulation of this crosstalk can lead to abnormal healing processes and is the foundation of many skin diseases. A relatively overlooked cell type that nevertheless plays critical roles in skin homeostasis, wound repair, and disease is the dendritic epidermal T cells (DETCs), which are also called γδT-cells. Given their varied roles in both physiological and pathological scenarios, interest in the regulation and function of DETCs has substantially increased. Moreover, their ability to regulate other immune cells has garnered substantial attention for their potential role as immunomodulators and in immunotherapies. In this article, we describe a protocol to isolate and culture DETCs and analyse them in vivo within the skin. These approaches will facilitate the investigation of their crosstalk with other cutaneous cells and the mechanisms by which they influence the status of the skin.


Graphic abstract:


Overall workflow to analyse DETCs in vitro and in vivo.


Keywords: Skin (皮肤), Wound healing (伤口愈合), Immune cells (免疫细胞), Dendritic epidermal cells (树突状表皮细胞), Proliferation (扩散), Histology (组织学), Immunohistochemistry (免疫组织化学)

Background

The skin is composed of multiple cell types, including keratinocytes, fibroblasts, and various immune cells, that work together to provide a physical and immune barrier against the external environment. One of the skin resident immune cells, dendritic epidermal T cells (DETCs), has been shown to play important roles in tissue homeostasis, repair, and pathophysiology. In the epidermis, DETCs exhibit a dendritic morphology that allows each cell to stay in physical contact with other epidermal cells such as keratinocytes and Langerhans cells (Jameson et al., 2002; Jameson and Havran, 2007). This intercellular communication between keratinocytes and Langerhans cells is mediated by factors that activate DETCs, which in turn modulate tissue homeostasis. For example, during wound healing, damaged and stressed keratinocytes express various antigens that are recognized by DETCs, resulting in their activation and release of various cytokines such as keratinocyte growth factor-1 and 2, insulin growth factor-1, and interleukin-2 (Gustafsson et al., 2020). In addition, DETCs also secrete fibroblast growth factor 9, which mediates hair neogenesis during wound healing (Gay et al., 2013). We recently uncovered a novel role of skin resident DETCs in regulating hair follicle stem cell activity in wounded skin (Lee et al., 2017). In addition, DETCs have also been shown to play an important role in maintaining the epidermis in unwounded skin as mice lacking DETCs exhibit higher levels of epidermal apoptosis (Sharp et al., 2005). In this protocol, we provide a detailed method for the isolation and culture of DETCs to investigate the effect of various soluble factors and cell-cell contacts on DETC activation and the downstream consequence on other cutaneous cells. We also describe a method for the in vivo analysis of DETCs, which will help in understanding the intercellular communication of DETCs at a tissue level in both physiological and pathological conditions.

Materials and Reagents

  1. 10 ml serological pipette (Stem Cell Technologies, catalog number: 38004)

  2. 70 µm cell strainer (Corning, catalog number: 431751)

  3. 10 cm Petri dish (Eppendorf, catalog number: 30702118)

  4. FACS tubes (Stem Cell Technologies, catalog number: 38007)

  5. 50 ml Falcon tube (Thermo Fisher, catalog number: 10788561)

  6. 15 ml Falcon tubes (Stem Cell Technologies, catalog number: 05860)

  7. Ultra-low attachment culture plates (Corning, catalog number: CLS3471-24EA)

  8. 96-well dish (Eppendorf, catalog number: EP0030730011-80EA)

  9. Kimwipes (Kimberly-Clark Kimtech Science, catalog number: 34155)

  10. C57BL6 mice (Jackson Laboratories)

  11. Ethanol (Sigma-Aldrich, catalog number: T4049 02870)

  12. Fetal Bovine Serum (Gibco, catalog number: 10270106)

  13. Phosphate Buffer Solution (PBS) (CSH Protocols, http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8247)

  14. Trypsin solution (Sigma-Aldrich, catalog number: T4049)

  15. E media without calcium (Nowak and Fuchs, 2009)

  16. 7-Aminoactinomycin D (7-AAD) (Thermo Fisher Scientific, catalog number: A1310)

  17. FITC isotype control (Thermo Fisher Scientific, catalog number: GM4992)

  18. PE-Cy7 isotype control (Thermo Fisher Scientific, catalog number: 25-4714 - 80)

  19. PE isotype control (Thermo Fisher Scientific, catalog number: 12-4714-42)

  20. FITC anti-Vγ3 TCR (Thermo Fisher Scientific, catalog number: MHGD01)

  21. PE-Cy7 anti-CD3ϵ (Thermo Fisher Scientific, catalog number: 25-0038-42)

  22. PE anti-γδTCR (Thermo Fisher Scientific, catalog number: 12-9959-42)

  23. Concanavalin A (Sigma-Aldrich, catalog number: C5275)

  24. Glutamine (Sigma-Aldrich, catalog number: G8540)

  25. HEPES (Sigma-Aldrich, catalog number: H3375-25G)

  26. Sodium pyruvate (Himedia, catalog number: PCT0503)

  27. NEAA (Merck, catalog number: 7145 M7145)

  28. Penicillin-Streptomycin (Merck, catalog number: P4333)

  29. β-Mercaptoethanol (ME) (Sigma-Aldrich, catalog number: M6250)

  30. Recombinant human IL-2 (Promo Cell, catalog number: 61241)

  31. Gentamicin (Thermo Fisher Scientific, catalog number: 15750078)

  32. RPMI-1640 medium (Thermo Fisher Scientific, catalog number: 11875101)

  33. Mouse IL-17 Quantikine ELISA Kit (R&D Systems, catalog number: M1700)

  34. FGF7 (Sigma-Aldrich, catalog number: RAB0188)

  35. TNFα (Thermo Fisher Scientific, catalog number: KHC3011)

  36. IFN-γ (Thermo Fisher Scientific, catalog number: RAB0223)

  37. IL-23 (Thermo Fisher Scientific, catalog number: PHC9321)

  38. Recombinant IL-1beta (10 ng/ml) (R&D Systems, catalog number: 201-LB-005/CF)

  39. DMEM/F-12, powder (Gibco, catalog number: 12500062)

  40. Sodium bicarbonate (Gibco, catalog number: S5761)

  41. Cholera toxin (Sigma-Aldrich, catalog number: C8052-.5MG)

  42. Hydrocortisone (Sigma-Aldrich, catalog number: H0888)

  43. Autoclaved Milli-Q distilled water

  44. Hydrochloric acid (Sigma-Aldrich, catalog number: 320331-500ML)

  45. Anti-CD3 (1 μg/ml) (Abcam, catalog number: 5690)

  46. Trypan blue (Thermo Fisher Scientific, catalog number: 15250061)

  47. Anti-JAML (Abcam, catalog number: 67843)

  48. WST-1 reagent (Merck, catalog number 5015944001)

  49. MTT reagent (Merck, catalog number: CT01-5)

  50. OCT medium (Thermo Scientific, catalog number: 23-730-571)

  51. 16% paraformaldehyde (Fisher Scientific, catalog number: 50-980-487)

  52. Triton X-100 (Thermo Scientific, catalog number: PI28313)

  53. Keratin 5 (Abcam, catalog number: ab52635)

  54. Anti-Ki67 (Abcam, catalog number: ab16667)

  55. Anti-γδTCR antibody (eBioscience, catalog number: 12-5711-82)

  56. Goat anti-Rabbit Alexa Fluor 488 (Molecular Probes, catalog number: A-11008)

  57. Goat anti-chicken Alexa Fluor 647 (Molecular Probes, catalog number: A-21449)

  58. DAPI (Abcam, catalog number: ab228549)

  59. Vectashield (Vector Laboratories, catalog number: H-1500)

  60. MOWIOL 4-88 Reagent (Sigma-Aldrich, catalog number: 475904-100GM-M)

  61. Tris (Sigma-Aldrich, catalog number: 10708976001)

  62. Glycerol (Sigma-Aldrich, catalog number: G5516)

  63. Superfrost Plus slides (VWR, catalog number: 48311-703)

  64. PBS with 2× antibiotics (see Recipes)

  65. E media without calcium (see Recipes)

  66. FACS Staining Buffer (see Recipes)

  67. RPMI media (see Recipes)

  68. 0.2% Triton X-100 (see Recipes)

  69. Blocking buffer for permeabilization (see Recipes)

  70. 4% Paraformaldehyde (PFA) (see Recipes)

  71. 70% Ethanol (see Recipes)

  72. Mowiol (see Recipes)

Equipment

  1. Fine forceps (Fisher Scientific, catalog number: NC9924848)

  2. Scissors (Fisher Scientific, catalog number: 08-951-20)

  3. Cell culture incubator (Eppendorf, model: EppendorfTM GalaxyTM 170)

  4. Aspirator (Sigma-Aldrich, catalog number: BMSV0020-1EA)

  5. FACS Aria (BD, model: FACSAriaTM III sorter)

  6. Centrifuge (Eppendorf, model: 5702)

  7. Spectrophotometer (Thermo ScientificTM GENESYSTM 20 Visible Spectrophotometer)

  8. Cell culture incubator (Eppendorf, model: EppendorfTM GalaxyTM 170 S)

  9. Cell counter (Thermo Fisher Scientific, model: Countess3)

  10. 7-17 DETC cell line (Boismenu and Havran, 1994)

  11. Cryostat (Leica, model: CM1950)

  12. -80°C freezer (Thermo Scientific, Forma Ultra-Low Temperature Upright DD Freezer)

  13. Hydrophobic pen (Merck, catalog number: Z377821-1EA)

  14. Humidifying chamber

  15. Compound Binocular Microscope (Celestron Labs, model: CB2000CF)

  16. Fluorescent microscope (Olympus, model: IX73), Confocal microscope FV 3000 5 laser (IEC60825-1:2007)

Software

  1. ImageJ version 1.46 software

  2. GraphPad Prism 6

Procedure

  1. Isolation, maintenance, and proliferation of DETCs

    Given the important roles of DETCs, methods to investigate the regulation and function of these cells are required. This is facilitated by the ability to isolate and establish primary cultures of DETCs and reconstitute their intercellular crosstalk with different cells in vitro.

    Isolation and culture of DETCs

    Isolation of DETCs from the skin via FACS (Kashem and Kaplan, 2018)

    1. Euthanize C57BL6 pups of postnatal days 0 to 5 via decapitation or a method approved by the Institutional Animal Ethics Committee. Three to four pups are required for a 3.5 cm dish of cultured DETCs.

    2. Clean the surface of the pup using 70% ethanol to decrease the chances of microbial contamination.

    3. Remove the limbs and tail of each pup as close to the core body as possible using sharp scissors. Insert the scissors through the hole made by the removal of the tail and cut the skin along the dorsal midline of the body all the way to the neck.

    4. Using forceps, grasp the skin and peel the whole skin off of the body, taking care not to tear the skin into pieces. Rinse the peeled skin by placing it in a tube with 10 ml of sterile PBS containing 2× antibiotics (see Recipe 1) for 10 min. Then remove excess PBS by blotting the skin on a tissue.

    5. In a new 10 cm Petri dish containing 10 ml of 0.25% trypsin, place the skin with the dermis side down making contact with the solution. Avoid submerging the epidermis in the trypsin solution to prevent over-digestion of the epidermis. Spread out the curled edges using fine forceps to maximize contact of the entire dermis with the trypsin solution.

    6. Incubate the skin in trypsin for one hour at 37°C or overnight at 4°C.


      Note: Steps A7 to A9 are performed at room temperature.

    7. Separate the epidermis from the dermis using fine forceps. The separated epidermis will appear as a thin opaque sheet. The dermis can be used to isolate dermal cells such as fibroblasts (Kashem and Kaplan, 2018). To view a detailed protocol and video of epidermal cells isolation, refer to Li et al. (2017). Cut the epidermis into small pieces with scissors. Transfer the epidermis and trypsin mixture to a new 50 ml tube.

      Using a 10 ml serological pipette, repeatedly pipette the mixture of epidermis and trypsin up and down to facilitate the dissociation of the tissue into individual cells. The serological pipette might get blocked due to clumps of tissue. Tap the pipette to remove the clumps from the pipette.

    8. Keep on pipetting until it becomes easy to pipette up and down without tapping. Pipette the solution around 10 to 15 times slowly to properly remove the clumps.

    9. Pass the cell suspension through a 70 µm cell strainer into a new 50 ml tube. Also, pass 5 ml of E media without calcium (see Recipe 2) through the cell strainer to remove any cells that are trapped in the strainer, which would also inactivate the trypsin.

    10. Centrifuge the cell suspension at 250 × g for 10 min in a swing bucket rotor at 4°C.

    11. Remove the supernatant carefully using an aspirator. Wash the cells once with PBS by resuspending the cell pellet in 5 ml of PBS and centrifuging at 250 × g for 5 min each at 4°C.

    12. Resuspend the cells in 1 ml of staining buffer (see Recipe 3) for FACS sorting.

      Note: For Staining Buffer, use chelated fetal bovine serum since the presence of calcium might lead to the formation of cell clumps.

    13. Count the number of viable cells using trypan blue staining (Figure 1).



      Figure 1. Preparation of cells for counting. Bright-field image of the cell suspension under the microscope for automated counting. Scale bar: 50 μm.


    14. To prevent the nonspecific binding of antibodies, incubate the cells with anti CD16/CD32 (1 µg/million cells) for 5 min at 4°C. CD16/CD32 incubation prevents nonspecific binding of immunoglobulins to FcγIII, FcγII, and possibly FcγI receptors.

    15. From this epidermal cell suspension, make five aliquots of 50 µl each in five different FACS tubes. Add 50 µl of staining buffer to each 50 µl aliquot to match the volumes listed in Table 1 and label samples as indicated.

      Note: At least 100,000 cells are required to calibrate the flow cytometer.


      Table 1. Summary of control and analyte samples required for FACS analysis. To calibrate the flow cytometer, various controls are required: unstained control (#1) and single stained and isotype controls (#2-5). For samples that are to be used to isolate DETCs (#6/analyte sample), cells are stained with all antibodies and resuspended in a staining buffer with 5 µl of 7AAD (Fluorochrome used to stain non-viable cells). Samples used for isolating DETCs might need to be diluted such that the event rate on the FACS machine is between 1,000-4,000 event/s (it is ideal to start with a higher concentration and dilute it later based on the event rate).


    16. After incubating the cells for 30 min in the dark, pellet the cells by centrifuging the tubes in a swing bucket rotor at 250 × g for 5 min at 4°C. Remove the supernatant carefully with an aspirator. Wash the cell pellet three times by resuspending it in 1 ml of staining buffer and centrifuging at 250 × g for 5 min at 4°C.

    17. DETCs can then be isolated using FACS, as explained in detail in Badarinath et al. (2019) and Nielsen et al. (2014).

      Other than primary DETCs, there is also a 7-17 DETC cell line, which can be used to study the effects of various factors on DETCs and their interactions with other cutaneous cells. The 7-17 cell line was originally established from FACS-purified DETC from AKR mice and expanded by repeated stimulation with concanavalin A (1 µg/ml) supplemented with rIL-2 (Edelbaum et al., 1995; Nielsen et al., 2014).

      Notes:

      1. Preparation of samples for flow cytometry and usage of the machine are complex processes and beyond the scope of this chapter. Before planning the FACS experiment, one should be familiar with the general background and theory of flow cytometry (Shapiro, 2003).

      2. For more details on the gating strategy while sorting, please refer to Wohn et al. (2014) and Havran et al. (1989).


  2. Culture of primary DETCs and 7-17 DETC cell line

    1. Both primary DETCs isolated by FACS and 7-17 DETC stable cell lines are cultured in RPMI 1640 media at 37°C, 5% CO2. The RPMI medium is supplemented with 10% FBS, 2 mM glutamine, 500 µl of Penicillin-Streptomycin (100×), 50 µM β-mercaptoethanol, 25 mM HEPES, 1 mM Na pyruvate, 100 µM nonessential amino acids, and 20 U/ml recombinant human IL-2 (Sharp et al., 2005; Nielsen et al., 2014).

    2. Primary DETCs and DETC cell lines can be cultured as suspension cultures by using ultra-low attachment culture plates. Every two days, remove half of the media by tilting the dish gently, allowing the cells to settle down and replenish with fresh media.

    3. Once cells become 70% confluent, passage them by collecting the total cell suspension into a 15 ml tube and centrifuge the cells at 300 × g for 5 min at room temperature. Remove the supernatant gently and resuspend the cells in 1 ml of fresh media. From this, add 200 µl to a new 10 cm dish containing 10 ml of fresh growth media and culture them in the same way as described above.

    4. In vitro cultures of DETCs are a useful platform to study their interactions with different cell types and the effect of various soluble factors. For example, DETCs can be activated by either cytokines secreted from neighbouring cells or by direct cell-cell interactions. It has also been observed that DETCs are activated by co-culturing them with hair follicle stem cells (Badarinath et al., 2019).

      Note: While culturing different cell types in co-culturing experiments, we should always be careful about the growth conditions of different cell types as inappropriate conditions for culturing any of the cell types can affect the cells in various ways such as stress, proliferation, and apoptosis.

    5. We can study the effect of various soluble secreted factors on the activation of DETCs. One method utilizes conditioned media from skin explants in which researchers examined the effect of IL-1α secreted from wounded keratinocytes on the activation of DETCs and is explained in detail in Lee et al. (2017) and Badarinath et al. (2019). Controls required for this experiment are DETCs treated with recombinant IL-1α and isolated DETCs from IL-1 receptor KO animals treated with IL-1α. The isolated cells can be utilized in experiments to determine the soluble and intercellular signals that affect both DETC behaviour (such as proliferation and activation) as well as its impact on neighbouring cells in the skin.

      Notes:

      1. For conditioning, use the same medium in which the subsequent culture experiments will be performed. Avoid serum in media for these experiments as serum will have its own effect on the cells.

      2. As an alternative control for DETC activation, these cells can be treated with recombinant IL-23 (10 ng/ml), recombinant IL-1beta (10 ng/ml), anti-CD3 (1 μg/ml), or combinations of these and scored for activation by measuring the expression of IL-17, FGF7, TNFα, and IFN-γ by ELISA or transcript levels as mentioned above.


  3. Proliferation assays for activated DETCs in vitro

    Elevated proliferation is one of the hallmarks of activated DETCs and can thus be used as a readout for activation. In addition to proliferation, other biomarkers of activated DETCs include the expression and secretion of various cytokines such as IL-17, FGF-7, TNFα, and IFN-γ. These secreted cytokines can be detected after 48 h using an ELISA kit (Nielsen et al., 2014) or are evident at the transcript levels after 24 h of treatment (Lee et al., 2017).


  4. Effect of secreted factors on DETCs

    1. For proliferation assays, culture DETCs as explained earlier. When cells are 70% confluent, collect the cells in a 15 ml Falcon tube and centrifuge at 300 × g for 5 min at room temperature.

    2. Dilute the control and test conditioned media 1:3 with fresh RPMI media.

      Note: Preparation of conditioned media is explained in detail in Badarinath et al. (2019).

    3. Resuspend the cells in 100 µl of respective conditioned media and, from those, plate 30,000 cells in each well of a 96-well dish.

    4. Incubate the cells in 100 µl of conditioned media for 24 to 48 h at 37°C.

    5. After incubation, remove the media containing DETCs and quantify proliferation at different time intervals such as 24, 48, and 72 h.

    There are various assays available by which you can count the number of cells for proliferation assays after treatment with various stimuli such as the MTT cell proliferation assay, WST-1 cell proliferation assay, and trypan blue cell counting.


  5. Effect of various factors secreted by DETCs on hair follicle stem cells

    Note: Isolate primary hair follicle stem cells as previously described in Nowak and Fuchs (2009).

    1. Treat DETCs with conditioned media from control or test skin/epidermal explants animal for 16-24 h.

    2. Collect the suspension culture in a 15 ml Falcon tube and centrifuge at 300 × g for 5 min. Collect the supernatant and discard the cell pellet.

    3. Dilute the conditioned media 1:5 with fresh E-media.

    4. Incubate hair follicle stem cells with diluted conditioned media for 24-48 h at 37°C, 7% CO2.

    5. Count the cells at different time intervals over the 24-48 h time period.


  6. In vivo analysis of DETC activation

    The tissue microenvironment strongly dictates the regulation and function of DETCs. Under homeostatic conditions, DETCs have a distinctive dendritic morphology, but after injury or stress, DETCs proximal to the wound site acquire a rounded morphology and transiently lose their dendrites. In addition to morphological changes, another marker of DETC activation is an increased proliferative index. Upon activation, DETCs release certain cytokines that play a significant role in maintaining the protective physical and immune barrier of the murine skin. A variation in DETC function can aggravate skin-related autoimmune diseases, impede tumour eradication, or disrupt proper wound healing (Cruz et al., 2018). Hence, in vivo analysis of DETCs advances our understanding of the function of these cells in both physiological conditions such as wound healing and a variety of pathological scenarios including fibrosis, inflammatory diseases, and carcinomas.

Data analysis

  1. Sectioning of mouse skin

    1. To embed skin in OCT medium, follow the protocol described in Gund et al. (2021).

    2. Section the frozen blocks as described in Fischer et al. (2008).

    3. Store the sections on the charged slides at -80°C.

      Notes:

      1. Collect the skin from the same region of the mice to compare the DETCs between the control and the test animal. It is known that there is heterogeneity of DETCs in different regions of the mouse skin.

      2. Since DETCs are dendritic in morphology when inactive, it is better to have thicker sections to visualize the morphology and quantify dendrites. Hence, take sections of10 µm thickness.


  2. Immunofluorescence assay for γδTCR and Ki67

    1. Remove the frozen slides with skin sections from the -80°C freezer and thaw them at room temperature for at least a minute (but not longer than 5 min).

    2. Using a kimwipe, carefully remove the condensation around the skin tissues. Place the slides in a humidifying chamber (Gund et al., 2021).

    3. Make a hydrophobic barrier around the tissue section using a hydrophobic pen. To fix the sections, add 50-100 µl of 4% PFA per tissue section for 10 min at room temperature. Make sure the tissue sections are completely covered with PFA.

      Notes:

      1. This allows you to minimise the volume of buffers and antibodies being used and gives the ability to differentially stain multiple skin sections on one slide.

      2. Make sure to read the antibody datasheet to utilize the appropriate fixative. This protocol is described for the antibodies mentioned in the reagents section.

      3. Do not exceed a fixation time of more than 10 min for tissues less than 10 µm thick as over-fixing leads to excess cross-linking of antigens and can produce false negative results.

      4. Thicker sections must be fixed for a longer time – An overnight incubation at 4°C is recommended.

    4. Aspirate the 4% PFA after 10 min of incubation. Wash the sections thoroughly with 50-100 µl of 1× PBS for 5 min each three times.

      Note: Aspiration can be avoided if the sections are loosely attached to the slide. Instead, one can remove the fixative/buffer using a pipette.

    5. Add enough blocking buffer to cover the tissue sections to block nonspecific interactions for 1 h at room temperature.

      Note: Freeze thawing of the section causes permeabilization of the plasma membrane and the integrity of the membrane proteins is maintained. Hence, additional permeabilization steps with any harsh detergents such as Tween-20 or Triton-X should be avoided as this can disrupt membrane proteins, especially if left for too long.

    6. Incubate the sections with primary antibody (γδTCR to mark DETCs and Ki67 to mark the proliferating cells) diluted in the blocking buffer (refer to table 2 for dilutions). Add 50-100 µl of the diluted primary antibody to each section and incubate overnight at 4°C in the humidifying chamber.

      Note: Make sure that the hydrophobic barrier is intact. If not, mark the boundaries again with a hydrophobic pen around the sections before adding the primary antibody.

    7. Aspirate the primary antibody and wash the sections with 1× PBS for 5 min each three times.

    8. Add 50-100 µl of secondary antibody (refer to Table 2 for the antibody dilutions) diluted in the blocking buffer onto the sections and incubate for 20 min at room temperature in the humidifying chamber.

      Note: γδTCR antibody used in this protocol is a conjugated antibody and does not require a secondary antibody.

    9. Aspirate the secondary antibody and wash the sections with 1× PBS for 5 min each three times.

    10. Mount the sections with Vectashield mounting media.

      Note: Sections can be mounted using alternative mounting media such as Mowiol or 80% glycerol.

    11. Image the slides under a fluorescent microscope.

      Note: Though the signal is optimal if imaged immediately after staining, the stained sections can be stored at 4°C before imaging the slides.

    12. Alternative method: If using other antibodies along with γδTCR that require additional permeabilization of the tissue, follow the procedure below:

      1. For staining of γδTCR, follow the protocol in section B up to step 8.

      2. Repeat steps B1 to B5 on the sections.

      3. Add 50 µl of permeabilization buffer (0.25% Triton X-100 in PBS) and incubate for 15 min at room temperature.

        Note: The concentration of Triton X-100 used for permeabilization should be determined for each antigen of interest.

      4. Block the sections with a blocking buffer containing goat serum and 0.1% Triton X-100 for 1 h at room temperature.

        Note: Blocking buffer should contain heat-inactivated normal serum from the same species as the host of the secondary antibody.

      5. Incubate the sections with primary antibody diluted in the blocking buffer for 2 h at room temperature.

      6. Follow steps B8 to B12 from section B of the protocol.


        Table 2. Summary of various primary and secondary antibodies used in section B along with their respective dilutions



  3. Imaging acquisition and analysis

    Image the stained sections under a fluorescent microscope. To quantify the number of proliferating DETCs, lower magnification images of 10× and 20× will suffice. To visualize the morphological differences between the inactive and active DETCs, higher magnification (>40×) images are required.

    Note: z-stack images are recommended to fully visualize the dendrites of DETCs that spread throughout the epidermis in multiple planes.

      Under homeostatic conditions, DETCs are generally inactive and restricted to the basal layer of the epidermis and the upper region of the hair follicle (Lee et al., 2009). As their name indicates, they possess a dendritic morphology. On the other hand, conditions in which DETCs are activated, such as a wound and mouse models of atopic dermatitis (Lee et al., 2009 and 2017) and squamous cell carcinoma (Du et al., 2010; De Craene et al., 2014), the cells lose their dendritic extensions and appear more rounded in morphology (Lee et al., 2009; Du et al., 2010). It has been previously reported that caspase 8 cKO mice, a model for atopic dermatitis (Lee et al., 2009; Du et al., 2010), exhibits activated DETCs (Lee et al., 2017). In this protocol, we demonstrate that this activation of DETCs (both morphological changes and increased proliferation) is conserved across mouse models with a strong inflammatory phenotype. For instance, we have observed that a mouse model for cutaneous squamous cell carcinoma (K14 Snail transgenic mice) (Du et al., 2010) also exhibits activation of DETCs as early as the neonatal stage (Figure 2 and Figure 3).



    Figure 2. Visualization of activated DETCs in WT and Snail transgenic skin. Immunofluorescence assay for Ki67 (green) to depict proliferation, γδTCR (red) to mark DETCs and K5 (blue) to mark the basal layer of epidermis on WT and SnTg skin section to observe the activation of DETCs. Scale bars: 50 µm.



    Figure 3. Morphological changes upon activation. Round morphology of DETCs marked by γδTCR (red) in Snail transgenic skin shows signs of activation compared to the dendritic morphology of DETCs in WT skin. Scale bars: 25 µm.


      When activated, DETCs proliferate and hence are Ki67 positive. Therefore, to quantify the number of proliferating DETCs, we counted the number of γδTCR positive cells that are positive for Ki67 across the epidermis of postnatal day 7 – WT and K14 Snail transgenic mice. The number of DETCs across the epidermis of WT and Snail transgenic skin is variable. Hence, to ensure consistency in quantification among different backgrounds, we counted all the γδTCR positive cells per 1 mm skin section.

      The percentage of DETCs that are Ki67 positive and negative can be quantified by using ImageJ as follows: Open the merged image of γδTCR and Ki67 on ImageJ software > Main menu > Plugin > Cell counter > Initialize > Type 1. Click on the cells that are positive for both γδTCR and Ki67. This will give the number of DETCs that are proliferating. To count the number of DETCs that are negative for Ki67, go to ImageJ software > Main menu > Plugin > Cell counter > Initialize > Type 2. The percentage of proliferating and non-proliferating DETCs can be calculated using the following formulae:

       


    The proportions of DETCs that are Ki67 positive and negative in WT and K14 Snail transgenic skin are as shown in Figure 4.



    Figure 4. Quantification of activated DETCs in WT and SnTg skin. Percentage plot depicting the proportion of proliferating DETCs, which suggests that Snail transgenic skin has more proliferating DETCs than WT skin.


      To quantify the circularity of the DETCs in WT and SnTg epidermis, ImageJ software is used. Taking the image of just the DETCs (γδTCR positive cells) > Mark the outline using the polygon selection tool on ImageJ for each DETC > Analyze > Set measurement > Area and shape description (select “Shape descriptor” parameter to measure and display) > Analyze > Measure or Ctrl+M. This will display a table with measurements. Perform this step for all DETCs in WT and SnTg skin sections. Circularity value of 1 indicates that a cell is a perfect circle, and values that approach 0 indicate that the cell is elongated. Here in this protocol, we have used a threshold circularity value of ≥ 0.5 to call a DETC active. A graph plotted using these measurements (Figure 5) shows that the DETCs in K14 Snail transgenic skin are more circular, indicating activation compared to that in the WT skin.



    Figure 5. Quantification of morphological changes observed in DETCs. Activated DETCs show morphological changes from a dendritic to circular shape, which were quantified using ImageJ software. Scatter plot shows that DETCs in Snail transgenic epidermis are more circular (circularity value ≥ 0.5) compared to DETCs in WT epidermis. Data represent the values ± SEM, ***P < 0.0001, based on the Student’s t-test.

Recipes

  1. PBS with 2× antibiotics

    Add 2 ml of Penicillin-Streptomycin (100×) to 10 ml of 10× PBS and bring volume up to 100 ml with ddH2O.

  2. E media without calcium

    Prepare E Media without Calcium for epidermal keratinocytes as described in Nowak and Fuchs (2009).

  3. FACS Staining Buffer

    2 ml of chelated Fetal Bovine Serum

    1 ml of Penicillin-Streptomycin (100×)

    100 µl of 50 mg/ml Gentamicin

    Add to 5 ml of 10× PBS

    Bring volume up to 50 ml with ddH2O

  4. RPMI media

    10% FBS

    2 mM glutamine

    500 µl of Penicillin-Streptomycin (100×)

    50 µM β-mercaptoethanol

    25 mM HEPES

    1 mM Na pyruvate

    100 µM nonessential amino acids

    20 U/ml recombinant human IL-2

    Bring volume up to 500 ml with RPMI media

  5. 0.2% Triton X-100

    Add 2 ml of 100% Triton X-100 to 100 ml of 10× PBS and bring volume up to 1 L with ddH2O.

  6. Blocking buffer for permeabilization

    Add 0.5 ml of goat serum (or serum originating from the same species as the secondary fluorescent conjugated antibody) and 10 µl of Triton X-100 in 1 ml of 10× PBS and bring the volume to 10 ml with ddH2O.

  7. 4% Paraformaldehyde (PFA)

    Dilute the 16% paraformaldehyde in 10× PBS by adding 12.5 ml of 16% of PFA to 5 ml of 10× PBS and bring the volume to 50 ml by adding ddH2O.

  8. 70% Ethanol

    Dilute 100% ethanol in distilled water by adding 700 ml of 100% ethanol in 300 ml distilled water.

  9. Mowiol

    1. Add 2.4 g of MOWIOL® 4-88 to 4.75 ml of glycerol and stir to mix.

    2. Add 6 ml of water and stir for several hours at room temperature.

    3. Add 12 ml of 0.2 M Tris (pH 8.5) and heat to 50°C for 10 min with occasional mixing.

    4. After the MOWIOL 4-88 dissolves, clarify the solution by centrifugation at 5,000 × g for 15 min.

Acknowledgments

The authors would like to thank Binita Dam and Johan Ajnabi for critical review of the manuscript. Work in the Jamora Laboratory is supported by inStem core funds and with past funding from the Department of Biotechnology of the Government of India (BT/PR8738/AGR/36/770/2013); the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH (5R01AR053185-03); and the American Cancer Society (15457-RSG-08-164-01-DDC). The animal work was partially supported by the National Mouse Research Resource (NaMoR) (BT/PR5981/MED/31/181/2012; 2013-2016) from the Department of Biotechnology. We thank the staff of the NCBS/inStem Animal Care and Resource Centre for assistance with animal husbandry, and the NCBS/inStem Central Imaging and Flow Cytometry Facility (CIFF) for help with image acquisition. We dedicate this protocol to the memory of our colleague and friend Professor Wendy Havran who was a pioneer and leader in the field of γδT-cell biology.

  This protocol was utilized in the following manuscript: Lee, P., Gund, R., Dutta, A., Pincha, N., Rana, I., Ghosh, S., Witherden, D., Kandyba, E., MacLeod, A., Kobielak, K., Havran, W. L. and Jamora, C. (2017). Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells. Elife 6: e28875.

Competing interests

No conflicts of interest to be declared.

Ethics

All work with animals was carried out at the NCBS/inStem Animal Care and Resource Centre and protocols were approved by the Institutional Animal Ethics Committee.

References

  1. Badarinath, K., Dutta, A., Hegde, A., Pincha, N., Gund, R. and Jamora, C. (2019). Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells. Methods Mol Biol 1879: 285-297.
  2. Boismenu, R. and Havran, W. L. (1994). Modulation of epithelial cell growth by intraepithelial γδ T cells. Science 266(5188): 1253-1255.
  3. Cruz, M. S., Diamond, A., Russell, A. and Jameson, J. M. (2018). Human αβ and γδ T Cells in Skin Immunity and Disease. Front Immunol 9: 1304.
  4. De Craene, B., Denecker, G., Vermassen, P., Taminau, J., Mauch, C., Derore, A., Jonkers, J., Fuchs, E. and Berx, G. (2014). Epidermal Snail expression drives skin cancer initiation and progression through enhanced cytoprotection, epidermal stem/progenitor cell expansion and enhanced metastatic potential. Cell Death Differ 21(2): 310-320.
  5. Du, F., Nakamura, Y., Tan, T. L., Lee, P., Lee, R., Yu, B. and Jamora, C. (2010). Expression of snail in epidermal keratinocytes promotes cutaneous inflammation and hyperplasia conducive to tumor formation. Cancer Res 70(24): 10080-10089.
  6. Edelbaum, D., Mohamadzadeh, M., Bergstresser, P. R., Sugamura, K. and Takashima, A. (1995). Interleukin (IL)-15 promotes the growth of murine epidermal γδ T cells by a mechanism involving the β- and γc-chains of the IL-2 receptor. J Invest Dermatol 105(6): 837-843.
  7. Fischer, A. H., Jacobson, K. A., Rose, J. and Zeller, R. (2008). Cryosectioning tissues. CSH Protoc 2008: pdb prot4991.
  8. Gay, D., Kwon, O., Zhang, Z., Spata, M., Plikus, M. V., Holler, P. D., Ito, M., Yang, Z., Treffeisen, E., Kim, C. D., Nace, A., Zhang, X., Baratono, S., Wang, F., Ornitz, D. M., Millar, S. E. and Cotsarelis, G. (2013). Fgf9 from dermal γδ T cells induces hair follicle neogenesis after wounding. Nat Med 19(7): 916-923.
  9. Gund, R., Zirmire, R., J, H., Kansagara, G. and Jamora, C. (2021). Histological and Immunohistochemical Examination of Stem Cell Proliferation and Reepithelialization in the Wounded Skin. Bio-protocol 11(2): e3894.
  10. Gustafsson, K., Herrmann, T. and Dieli, F. (2020). Editorial: Understanding γδ T Cell Multifunctionality - Towards Immunotherapeutic Applications. Front Immunol 11: 921.
  11. Havran, W. L., Grell, S., Duwe, G., Kimura, J., Wilson, A., Kruisbeek, A. M., O'Brien, R. L., Born, W., Tigelaar, R. E. and Allison, J. P. (1989). Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody. Proc Natl Acad Sci U S A 86(11): 4185-4189.
  12. Shapiro, H. M. (2003). Practical Flow cytometry. John Wiley & Sons, Inc.
  13. Jameson, J. and Havran, W. L. (2007). Skin γδ T-cell functions in homeostasis and wound healing. Immunol Rev 215: 114-122.
  14. Jameson, J., Ugarte, K., Chen, N., Yachi, P., Fuchs, E., Boismenu, R. and Havran, W. L. (2002). A role for skin γδ T cells in wound repair. Science 296(5568): 747-749.
  15. Kashem, S. W. and Kaplan, D. H. (2018). Isolation of Murine Skin Resident and Migratory Dendritic Cells via Enzymatic Digestion. Curr Protoc Immunol 121(1): e45.
  16. Lee, P., Lee, D. J., Chan, C., Chen, S. W., Ch'en, I. and Jamora, C. (2009) Dynamic expression of epidermal caspase 8 simulates a wound healing response. Nature 458(7237):519-23.
  17. Lee, P., Gund, R., Dutta, A., Pincha, N., Rana, I., Ghosh, S., Witherden, D., Kandyba, E., MacLeod, A., Kobielak, K., Havran, W. L. and Jamora, C. (2017). Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells. Elife 6: e28875.
  18. Li, F., Adase, C. A. and Zhang, L. J. (2017). Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin. J Vis Exp (125): 56027.
  19. Nielsen, M. M., Lovato, P., MacLeod, A. S., Witherden, D. A., Skov, L., Dyring-Andersen, B., Dabelsteen, S., Woetmann, A., Odum, N., Havran, W. L., Geisler, C. and Bonefeld, C. M. (2014). IL-1beta-dependent activation of dendritic epidermal T cells in contact hypersensitivity. J Immunol 192(7): 2975-2983.
  20. Nowak, J. A. and Fuchs, E. (2009). Isolation and culture of epithelial stem cells. Methods Mol Biol 482: 215-232.
  21. Sharp, L. L., Jameson, J. M., Cauvi, G. and Havran, W. L. (2005). Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1. Nat Immunol 6(1): 73-79.
  22. Wohn, C. T., Pantelyushin, S., Ober-Blobaum, J. L. and Clausen, B. E. (2014). Aldara-induced psoriasis-like skin inflammation: isolation and characterization of cutaneous dendritic cells and innate lymphocytes. Methods Mol Biol 1193: 171-185.

简介

[摘要]皮肤在保护身体免受外界环境中的病原体和化学物质侵害方面起着重要作用。受伤后,愈合程序会迅速启动,并涉及广泛的细胞间通讯以恢复组织稳态。这种串扰的放松管制会导致异常的愈合过程,并且是许多皮肤病的基础。树突状表皮 T 细胞 (DETC),也称为γδ T 细胞,但在皮肤稳态、伤口修复和疾病中起着关键作用的一种相对被忽视的细胞类型。鉴于它们在生理和病理场景中的不同作用,对 DETC 的调节和功能的兴趣大大增加。此外,它们调节其他免疫细胞的能力因其作为免疫调节剂和免疫疗法的潜在作用而备受关注。在这篇文章中,我们描述了一个协议,分离,培养DETCs并分析它们在体内的皮肤内。这些方法将有助于研究它们与其他皮肤细胞的串扰以及它们影响皮肤状态的机制。


图文摘要:

整体工作流程ANALY小号ÈDETCs体外和体内。


[背景]皮肤由多种细胞类型的,includ荷兰国际集团的角质形成细胞,成纤维细胞,和各种免疫细胞,一起工作,以提供一个相对于外部环境的物理和免疫屏障。一个皮肤免疫居民细胞,树突状表皮T细胞(DETCs)的,已被证明在组织稳态,修复中发挥重要作用,与病理生理学。在表皮,DETCs表现出树枝状形态,其允许每个单元留在与其他表皮细胞如角质形成细胞和郎格罕物理接触小号细胞(詹姆森等人,2002 ;詹姆森和哈夫兰,2007)。角质形成细胞和之间的这种细胞间通讯朗格汉斯细胞是通过介导因素即激活DETCs,这反过来调制组织动态平衡。例如,在伤口愈合过程中,受损和强调角质形成细胞表达由DETCs识别各种抗原,从而导致它们的活化和各种细胞因子如角质形成细胞生长因子1和2,胰岛素生长因子-1和白细胞介素2的释放( Gustafsson等人,2020 年)。此外,DETC 还分泌成纤维细胞生长因子 9,其在伤口愈合过程中介导毛发新生(Gay等,2013)。我们[R ecently发现皮肤居民DETCs的在创伤皮肤调节毛囊干细胞活性的新角色(李等人,2017年)。此外,由于缺乏 DETCs 的小鼠表现出更高水平的表皮细胞凋亡,DETCs 也已被证明在维持未受伤皮肤的表皮方面发挥重要作用(Sharp等,2005)。在这个协议中,我们提供的详细方法为分离和文化性Ë DETCs的调查对DETC激活和其他皮肤细胞中的下游后果各种可溶性因子和细胞-细胞接触的效果。我们还描述了一种对DETCs进行体内分析的方法,这将有助于了解 DETCs 在生理和病理条件下在组织水平上的细胞间通讯。

关键字:皮肤, 伤口愈合, 免疫细胞, 树突状表皮细胞, 扩散, 组织学, 免疫组织化学


材料和试剂

 
10毫升血清移液管(干ç ELL Ť echnologies,目录号:38004)
70 µm 细胞过滤器(Corning,目录号:431751)
10 cm培养皿(Eppendorf,目录号:30702118)
FACS管(干ç ELL Ť echnologies,目录号:38007)
50毫升Falcon管(热˚F isher,目录号:10788561)
15毫升Falcon管(干ç ELL Ť echnologies,目录号:05860)
超低附着培养板(Corning,目录号:CLS3471-24EA)
96 - 孔盘(Eppendorf,目录号:EP0030730011-80EA)
Kimwipes(Kimberly-Clark Kimtech Science,目录号:34155 )
C57BL6 小鼠(Jackson L aboratories)
乙醇(Sigma - Aldrich,目录号:T4049 02870)
胎牛血清(Gibco,目录号:10270106)
磷酸缓冲液(PBS)(CSH P rotocols,http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8247)
胰蛋白酶溶液(Sigma - Aldrich,目录号:T4049)
不含钙的 E 培养基(Nowak和Fuchs,2009 年)
7-Aminoactin omycin D(7-AAD)(Thermo Fisher Scientific,目录号:A1310)
FITC 同种型对照(Thermo Fisher Scientific,目录号:GM4992)
PE-Cy7同种型对照(Thermo Fisher Scientific,目录号:25-4714-80)
PE同种型对照(Thermo Fisher Scientific,目录号:12-4714-42)
FITC 抗 Vγ3 TCR(Thermo Fisher Scientific,目录号:MHGD01)
PE-Cy7抗CD3ε(Thermo Fisher Scientific,目录号:25-0038-42)
PE抗γδTCR(Thermo Fisher Scientific,目录号:12-9959-42)
Concanavalin A(Sigma - Aldrich,目录号:C5275)
谷氨酰胺(Sigma - Aldrich,目录号:G8540)
HEPES(Sigma - Aldrich,目录号:H3375-25G)
丙酮酸钠(Himedia,目录号:PCT0503)
NEAA(默克,目录号:7145 M7145)
青霉素-链霉素(Merck,目录号:P4333)
β-巯基乙醇(ME)(Sigma - Aldrich,目录号:M6250)
重组人IL-2(促销ç ELL,目录号:61241)
庆大霉素(Thermo Fisher Scientific,目录号:15750078)
RPMI-1640 培养基(Thermo Fisher Scientific,目录号:11875101)
小鼠IL-17的Quantikine ELISA试剂盒(R&d小号ystems,目录号:M1700)
FGF7(Sigma - Aldrich,目录号:RAB0188)
TNFα(Thermo Fisher Scientific,目录号:KHC3011)
IFN-γ(Thermo Fisher Scientific,目录号:RAB0223)
IL-23(Thermo Fisher Scientific,目录号:PHC9321)
重组IL-1β(10纳克/毫升)(R&d小号ystems,目录号:201-LB-005 / CF)
DMEM/F-12,粉末(Gibco,目录号:12500062)
碳酸氢钠(Gibco,目录号:S5761)
霍乱毒素(Sigma - Aldrich,目录号:C8052-.5MG)
氢化可的松(Sigma - Aldrich,目录号:H0888)
蒸压中号ILLI-Q蒸馏水
盐酸(Sigma - Aldrich,目录号:320331-500ML)
抗 CD3(1 μg/ml)(Abcam,目录号:5690)
台盼蓝(Thermo Fisher Scientific,目录号:15250061)
抗 JAML(Abcam,目录号:67843)
WST-1 试剂(默克,目录号 5015944001)
MTT试剂(默克,目录号:CT01-5)
OCT培养基(Thermo Scientific ,目录号:23-730-571)
16%多聚甲醛(费舍尔小号系统求解,目录号:50-980-487)
Triton X-100(Thermo Science ,目录号:PI28313)
角蛋白 5(Abcam,目录号:ab52635)
Anti-Ki67(Abcam,目录号:ab16667)
抗γδTCR抗体(eBioscience,目录号:12-5711-82)
山羊抗兔Alexa氟488(分子P长袍,目录号:A-11008 )
山羊抗鸡的Alexa氟647(分子P长袍,目录号:A-21449 )
DAPI(Abcam,目录号:ab228549)
Vectashield(Vector L aboratories,目录号:H-1500)
MOWIOL 4-88 试剂(Sigma - Aldrich,目录号:475904-100GM-M)
Tris(Sigma - Aldrich,目录号:10708976001)
甘油(Sigma - Aldrich,目录号:G5516)
Superfrost P lus幻灯片(VWR,目录号:48311-703)
PBS用2 ×抗生素(见ř ecipes)
e媒体无钙(见ř ecipes)
FACS染色缓冲液(见ř ecipes)
RPMI培养基(见ř ecipes)
0.2%的Triton X-100 (见ř ecipes)
封闭缓冲液透(见ř ecipes)
4%多聚甲醛(PFA) (见ř ecipes)
70% 乙醇(见配方)
Mowiol (见食谱)
 
设备
 
细镊子(Fisher Scientific ,目录号:NC9924848)
剪刀(Fisher Scientific ,目录号:08-951-20)
细胞培养箱(Eppendorf,型号:Eppendorf TM Galaxy TM 170 )
吸气器(Sigma - Aldrich,目录号:BMSV0020-1EA)
FACS Aria(BD ,型号:FACSAria TM III 分选机)
离心机仪(Eppendorf,米Odel等:5702)
分光光度计(Thermo Scientific TM GENESYS TM 20 可见分光光度计)
细胞培养箱(Eppendorf,型号:Eppendorf TM Galaxy TM 170 S)
细胞计数器(Thermo Fisher Scientific ,型号:Countess3)
7-17 DETC 细胞系(Boismenu和Havran,1994)
低温恒温器(徕卡,型号:CM1950)
-80 °C冷冻机(Thermo Scientific,Forma 超低温立式 DD 冷冻机)
疏水笔(默克,目录号:Z377821-1EA)
加湿室
化合物双目显微镜(塞莱斯特实验室,米Odel等:CB2000CF)
荧光显微镜(Olympus,型号:IX73),共聚焦显微镜 FV 3000 5 激光(IEC60825-1:2007)
 
软件
 
ImageJ 1.46版软件
GraphPad棱镜6
 
程序
 
DETC 的分离、维护和增殖
鉴于 DETC 的重要作用,需要研究这些细胞的调节和功能的方法。这得益于分离和建立 DETC 原代培养物的能力,并在体外重建它们与不同细胞的细胞间串扰。
分离和文化性Ë DETCs的
通过 FACS 从皮肤中分离 DETC(Kashem 和 Kaplan,2018 年)
产后天安乐死C57BL6幼仔小号通过断头或0至5的方法核定由机构动物伦理委员会。培养 DETC 的 3.5 厘米培养皿需要三到四只幼崽。
使用 70% 乙醇清洁幼崽的表面,以减少微生物污染的机会。
使用锋利的剪刀将每只小狗的四肢和尾巴尽可能靠近核心身体。将剪刀插入通过去除尾部形成的孔,沿着身体的背中线一直剪到颈部的皮肤。
使用镊子,把握皮肤和剥离整个皮肤脱去了身上的,注意不要撕破皮肤成片。将去皮的皮肤放入装有 10 ml 含有 2 ×抗生素(参见配方1)的无菌 PBS的管中 10 分钟,以冲洗去皮。然后通过在组织上涂抹皮肤来去除多余的 PBS。
在一个新的10厘米P含有10ml 0.25%胰蛋白酶的ETRI盘,放置与皮肤的真皮面朝下使与所述溶液接触。避免在胰蛋白酶溶液,以防止过度淹没表皮-表皮的消化。使用细镊子展开卷边,以最大限度地使整个真皮与胰蛋白酶溶液接触。
在 37 °C 下在胰蛋白酶中孵育皮肤一小时或在 4 °C 下过夜。
 
注意:步骤 A7 到 A9 在室温下进行。
使用细镊子将表皮与真皮分开。分离的表皮将显示为薄的不透明片。真皮可用于分离真皮细胞,例如成纤维细胞(Kashem 和 Kaplan,2018 年)。要查看表皮细胞分离的详细协议和视频,请参阅 Li等人。( 2017)。用剪刀将表皮剪成小块。将表皮和胰蛋白酶混合物转移到新的 50 毫升管中。
使用 10 ml 血清移液管,反复吸取表皮和胰蛋白酶的混合物,以促进组织分离成单个细胞。由于组织团块,血清移液管可能会被堵塞。轻敲移液器以从移液器中取出团块。
继续移液,直到无需敲击即可轻松上下移液。缓慢吸取溶液 10 到 15 次,以正确去除团块。
将细胞悬液通过 70 µm 细胞过滤器放入新的 50 ml 管中。此外,通过5毫升e媒体的无钙(见配方2)通过细胞过滤器以去除被捕获在straine任何细胞- [R ,这也将在激活所述胰蛋白酶。
在 4 °C 下,将细胞悬液在摆斗转子中以 250 × g离心10 分钟。
使用抽吸器小心地取出上清液。通过重悬细胞沉淀在5ml PBS和离心机的洗涤细胞一次,用PBS荷兰国际集团在250 ×克,每次5分钟,在4 ℃下。
用 1 ml 染色缓冲液(参见配方3)重悬细胞以进行 FACS 分选。
注意:对于染色缓冲液,请使用螯合胎牛血清,因为钙的存在可能会导致细胞团块的形成。
使用台盼蓝染色计算活细胞的数量(图 1)。
 
 
图 1. 用于计数的细胞的制备。亮-显微镜进行自动计数下将细胞悬浮液的场图像。比例尺:50μ米。
 
为了防止抗体的非特异性结合,在 4 °C下用抗 CD16/CD32(1 µg/百万细胞)孵育细胞 5 分钟。CD16 / CD32温育非特异性阻止免疫球蛋白到FcγIII,FcγII的结合,以及可能FcγI受体。
从这个表皮细胞悬液中,在五个不同的 FACS 管中制作五份 50 µl 的等分试样。将 50 µl 染色缓冲液添加到每 50 µl 等分试样中,以匹配表 1 中列出的体积并按指示标记样品。
注意:校准流式细胞仪至少需要 100,000 个细胞。
 
表1 。FACS 分析所需的对照和分析物样品汇总。要校准流式细胞仪,需要各种控制:未染色控制 (#1) 和单染色和同种控制 (#2-5)。对于样品的是要被用于分离DETCs(#6 /分析物的样品),细胞被染色的所有抗体,再悬浮于染色缓冲液机智H 5微升7AAD的(荧光染料用于染色非活细胞)。用于分离DETCs可能需要的样品进行稀释,使得对事件速率的FACS机器1之间,000-4 ,000事件/秒(它是理想的开始与一个更高浓度和更高稀释基于所述事件发生率)。
 
将细胞在黑暗中孵育 30 分钟后,通过在 4 °C 下以 250 × g的摆斗转子将管离心5 分钟来沉淀细胞。用抽吸器小心地取出上清液。将细胞沉淀重悬在 1 ml 染色缓冲液中,并在 4 °C 下以 250 × g离心5 分钟,将细胞沉淀洗涤 3 次。
然后可以使用 FACS 分离 DETC ,如Badarinath等人详细解释的那样。(2019 年)和尼尔森等人。(2014)。
比主DETCs其他,也有一个7-17 DETC细胞系,其可以被用于研究效果š对DETCs各种因素和它们的相互作用小号与其他皮肤细胞。在7-17细胞系最初从FACS建立-从AKR小鼠纯化DETC并通过反复刺激伴刀豆球蛋白A补充有的rIL-2((1微克/毫升)膨胀Edelbaum等人,1995;尼尔森等人,2014 ) 。
笔记:
用于流cytomet样品的制备RY和机器的使用是复杂的过程,ES和超出了本章的范围。在计划 FACS 实验之前,应熟悉流式细胞术的一般背景和理论(Shapiro, 2003) 。
有关排序时门控策略的更多详细信息,请参阅Wohn等人。( 2014 ) 和Havran 等人。( 1989) 。
 
原代 DETC 和 7-17 DETC 细胞系的培养
由 FACS 分离的原代 DETC 和 7-17 DETC 稳定细胞系均在 RPMI 1640 培养基中培养,温度为 37 °C 、5% CO 2 。在RPMI培养基中补充有10%FBS,2mM谷氨酰胺,500微升青霉素-链霉素(100 × ),50 μ中号β巯基乙醇,25mM的HEPES,1mM的丙酮酸钠,100μM非必需氨基酸,和20U /ml重组人IL-2(Sharp等人,2005 年;Nielsen等人,2014 年)。
初级DETCs和DETC细胞系可以作为悬浮培养物中培养小号通过使用超低附着培养板中。每两天,轻轻倾斜培养皿,取出一半培养基,让细胞安定下来并补充新鲜培养基。
一旦细胞达到 70% 汇合,通过将总细胞悬浮液收集到 15 ml 管中并在室温下以 300 × g离心细胞5 分钟来传代。轻轻取出上清液,将细胞重悬在 1 ml 新鲜培养基中。由此,将 200 µl 添加到一个新的 10 cm 培养皿中,该培养皿含有 10 ml 新鲜生长培养基,并以上述相同的方式培养它们。
DETCs 的体外培养是研究它们与不同细胞类型的相互作用和各种可溶性因子的影响的有用平台。例如,DETCs 可以被邻近细胞分泌的细胞因子激活,也可以被细胞间的直接相互作用激活。还观察到 DETC 通过与毛囊干细胞共培养而被激活(Badarinath等,2019)。
注:虽然在共培养实验中培养不同类型的细胞,我们要时刻注意对不同的细胞类型为不适当条件下的生长条件为培养任何细胞类型可以影响以各种方式,如紧张,增殖的细胞,和细胞凋亡。
我们可以研究各种可溶性分泌因素对效果的DETCs的激活。一种方法是利用从皮肤外植体在该研究者检查从受伤的角质形成细胞分泌的上IL-1α的效果条件培养基的DETCs的活化并详细描述于利说明等。( 2017 ) 和Badarinath等人。(2019)。该实验需要控制被DETCs用重组IL-1α处理的和分离DETCs从IL-1受体KO动物小号用IL-1α处理。分离出的细胞可用于实验以确定影响 DETC行为(如增殖和激活)及其对皮肤中邻近细胞的影响的可溶性和细胞间信号。
笔记:
对于调理,使用将在其中进行后续培养实验的相同培养基。在这些实验中避免在培养基中使用血清,因为血清会对细胞产生其自身的影响。
作为 DETC 激活的替代对照,这些细胞可以用重组 IL-23 (10 ng/ml)、重组 IL-1beta (10 ng/ml)、抗 CD3 (1 μg/ml) 或这些的组合进行处理并通过测量IL-17,FGF7,TNFα的表达得分为激活,如上所述通过ELISA或转录物水平和IFN-γ。
 
体外活化 DETC 的增殖试验
增殖增加是激活的 DETC 的标志之一,因此可用作激活的读数。除增殖外,活化的 DETC 的其他生物标志物还包括各种细胞因子的表达和分泌,例如 IL-17、FGF-7、TNFα 和 IFN-γ。这些分泌的细胞因子可以在 48 小时后使用 ELISA 试剂盒进行检测(Nielsen等,2014),或者在处理 24 小时后在转录水平上很明显(Lee等,2017)。
 
                   分泌因子对 DETCs 的影响
对于增殖分析,如前文所述,培养 DETC。当细胞是70%汇合,收集在细胞中一个15毫升˚F爱尔康管和离心机在300 ×克,在室温下5分钟。
用新鲜的 RPMI 培养基稀释控制和测试条件培养基 1:3。
注意:Badarinath 等人详细解释了条件培养基的制备。(2019)。
将细胞重悬于100微升各自的条件培养基和,从那些,板中的每个30,000细胞孔的96 -孔培养皿。
将细胞在 100 µl 条件培养基中在 37 °C下孵育24 至 48 小时。
温育后,除去含DETCs和量化增殖媒体在不同的时间间隔如24,48 ,和72小时。
有可用的多种测定法,通过它可以计数的细胞数为增殖测定小号与各种刺激物如处理后的MTT细胞增殖测定法,WST-1细胞增殖测定法,和台盼蓝细胞计数。
 
DETCs分泌的各种因子对毛囊干细胞的影响
注意:如之前 Nowak 和 Fuchs ( 2009)所述,分离原代毛囊干细胞。
用来自对照或测试皮肤/表皮外植体动物的条件培养基处理 DETCs 16-24 小时。
将悬浮培养物收集在 15 ml F alcon 管中,并以 300 × g离心5 分钟。收集上清液并丢弃细胞沉淀。
用新鲜的电子媒体以 1:5 的比例稀释条件培养基。
用稀释的条件培养基在 37 °C 、7% CO 2下孵育毛囊干细胞 24-48 小时。
在 24-48 小时的时间段内以不同的时间间隔计算细胞。
 
DETC 活化的体内分析
组织微环境强烈决定了 DETC 的调节和功能。在稳态条件下,DETCs 具有独特的树突形态,但在受伤或压力后,靠近伤口部位的 DETCs 获得圆形形态并暂时失去其树突。除形态学变化外,DETC 激活的另一个标志是增殖指数增加。激活后,DETC 会释放某些细胞因子,这些细胞因子在维持小鼠皮肤的保护性物理和免疫屏障方面发挥着重要作用。DETC 功能的变化会加重与皮肤相关的自身免疫性疾病,阻碍肿瘤根除,或破坏正常的伤口愈合(Cruz等人,2018 年)。因此,对 DETC 的体内分析促进了我们对这些细胞在生理条件(如伤口愈合)和各种病理情况(包括纤维化、炎症性疾病和癌)中的功能的理解。
 
数据分析
 
小鼠皮肤切片
要将皮肤嵌入 OCT 介质中,请遵循 Gund等人中描述的协议。(2021)。
按照 Fischer等人的描述对冷冻块进行切片。(2008 年)。
将切片存放在 -80 °C的带电幻灯片上。
笔记:
从小鼠的同一区域收集皮肤,以比较对照和试验动物之间的 DETC。众所周知,小鼠皮肤不同区域的 DETCs 存在异质性。
由于 DETC 在不活动时在形态上是树突状的,因此最好有更厚的部分来可视化形态和量化树突。因此,取部分≥ 10米μ微米厚。
 
γδTCR 和 Ki67 的免疫荧光检测 
从 -80 °C冰箱中取出带有皮肤切片的冷冻载玻片,并在室温下解冻至少一分钟(但不超过 5 分钟)。
使用无尘纸,小心地取出皮肤组织周围的凝结。将载玻片放入加湿室(Gund等人,2021 年)。
使用疏水笔在组织切片周围制作疏水屏障。为了固定段,加50-100 μ 4%PFA的升每组织切片在室温下10分钟。确保组织切片完全被 PFA 覆盖。
注意小号:
这使您可以最大限度地减少使用的缓冲液和抗体的体积,并能够对一张载玻片上的多个皮肤切片进行差异染色。
请务必阅读抗体数据表以使用适当的固定剂。该协议针对试剂部分中提到的抗体进行了描述。 
不超过一个固定的时间的10分钟以上的组织为小于10 μ微米厚作为过定影导致过量交-抗原连接,并且能够产生假阴性结果。
较厚的部分必须固定更长时间 –建议在 4 °C 下孵育过夜。
孵育 10 分钟后吸出 4% 的粉煤灰。用50-100彻底清洗部μ 1升× PBS,每次5分钟三次。
注意:如果切片松散地附着在载玻片上,则可以避免吸入。相反,可以使用移液器去除固定剂/缓冲液。
添加足够的封闭缓冲液以覆盖组织切片,以在室温下阻止非特异性相互作用 1 小时。
注意:切片的冻融会导致质膜透化,并保持膜蛋白的完整性。因此,应避免使用任何苛刻的去污剂(如 Tween-20 或 Triton-X)进行额外的透化步骤,因为这会破坏膜蛋白,特别是如果放置时间过长。 
孵育在封闭缓冲液稀释,并用一级抗体(γδTCR标记DETCs和Ki67标记增殖细胞)的部分(参照到表2稀释)。添加50-100 μ稀释的初级抗体的升向每个部分,并培育过夜,在4 ℃下在加湿室中。
注意:确保疏水屏障完好无损。如果没有,再与标记的边界一前加入所述部分周围的疏水性笔的初级抗体。
吸出一抗,每 3 次用 1 × PBS清洗切片5 分钟。
添加50-100 μ二级抗体的升(参照到Ť能够2用于抗体稀释液)在封闭缓冲液中稀释到部分并孵育在加湿室中室温下20分钟。
注意:本协议中使用的 γδTCR 抗体是偶联抗体,不需要二抗。
             吸出二抗,每 3 次用 1 × PBS清洗切片5 分钟。
          使用 Vectashield 安装介质安装部分。
注意:可以使用替代安装介质(例如 Mowiol 或 80% 甘油)安装切片。
在荧光显微镜下对幻灯片进行成像。
注意:虽然染色后立即成像信号是最佳的,但染色切片可以在 4°C 下保存,然后再对载玻片成像。
替代方法:如果使用其他抗体以及需要对组织进行额外透化的 γδTCR,请按照以下步骤操作:
对于 γδTCR 的染色,请按照 B 部分中的协议进行操作,直至步骤 8。
对切片重复步骤B 1 到B 5。
添加50 μ透化缓冲液(0.25%的Triton X-100的PBS),并培育的升,在室温下15分钟。
注意:用于透化的 Triton X-100 的浓度应针对每种感兴趣的抗原确定。
用含有山羊血清和 0.1% Triton X - 100的封闭缓冲液在室温下封闭切片1 小时。
注意:封闭缓冲液应包含与二抗宿主相同物种的热灭活正常血清。
在室温下用在封闭缓冲液中稀释的初级抗体孵育切片 2 小时。
按照协议B部分中的步骤B 8 到B 12。
 
表2 。B 部分中使用的各种一抗和二抗及其各自的稀释度的总结
 
影像采集与分析
在荧光显微镜下对染色切片进行成像。为了量化增殖的 DETC 的数量,10 ×和 20 × 的较低放大率图像就足够了。为了可视化非活动和活动 DETC 之间的形态差异,需要更高放大率 (>40 × ) 的图像。
注意:建议使用 z 堆栈图像来完全可视化在多个平面中遍布整个表皮的 DETC 的树突。
  在稳态条件下,DETCs 通常是无活性的并且局限于表皮的基底层和毛囊的上部区域(Lee等,2009)。顾名思义,它们具有树枝状形态。另一方面,DETC 被激活的条件,例如特应性皮炎(Lee等人,2009 年和2017 年)和鳞状细胞癌(Du等人,2010 年;De Craene等人,2014),细胞失去了它们的树突状延伸,并且在形态上看起来更圆(Lee et al. , 2009; Du et al. , 2010)。之前曾报道过半胱天冬酶 8 cKO 小鼠,一种特应性皮炎模型(Lee等人,2009 年;Du等人,2010 年),表现出激活的 DETC(Lee等人,2017 年)。在这个协议中,我们证明了这种 DETCs 的激活(形态变化和增殖增加)在具有强烈炎症表型的小鼠模型中是保守的。例如,我们观察到皮肤鳞状细胞癌的小鼠模型(K14 Snail 转基因小鼠)(Du等人,2010 年)早在新生儿阶段也表现出 DETC 的激活(图 2 和图 3)。
 
 
图 2. WT 和Snail 转基因皮肤中激活的 DETC 的可视化。Ki67(绿色)的免疫荧光分析用于描述增殖,γδTCR(红色)用于标记 DETC,K5(蓝色)用于标记 WT 和 SnTg 皮肤切片上的表皮基底层,以观察 DETC 的激活。比例尺小号:50 μ米。
 
 
图 3. 激活后的形态变化。与 WT 皮肤中 DETC 的树突形态相比,Snail转基因皮肤中由 γδTCR(红色)标记的 DETC 的圆形形态显示出激活的迹象。比例尺s :25 µm 。
 
  当被激活时,DETC 会增殖,因此 Ki67 呈阳性。因此,为了量化增殖的 DETC 的数量,我们计算了出生后第 7 天- WT 和 K14 Snail 转基因小鼠表皮中 Ki67 阳性的 γδTCR 阳性细胞的数量。WT 和 Snail转基因皮肤表皮上的 DETC 数量是可变的。因此,为了确保不同背景之间量化的一致性,我们计算了每 1毫米皮肤切片的所有 γδTCR 阳性细胞。
  在p是Ki67阳性和阴性DETCs的ercentage可以通过使用ImageJ如下量化:打开γδTCR和Ki67的合并图像上ImageJ软件>主菜单>插件>细胞计数器>初始化>类型1.点击细胞γδTCR和 Ki67均为阳性。这将给出正在增殖的 DETC 的数量。计数是阴性的Ki67 DETCs的数目,去ImageJ软件>主菜单>插件>细胞计数器>初始化>类型2在P增殖和非增殖DETCs的ercentage可以使用下列公式计算:
 
比例š DETCs的那是Ki67阳性和在WT和K14蜗牛负转基因皮肤如示于图4。
 
 
图 4. WT 和 SnTg 皮肤中激活的 DETC 的量化。描述增殖 DETC 比例的百分比图,这表明 Snail 转基因皮肤比WT 皮肤具有更多增殖的 DETC 。
 
  为了量化WT和SnTg表皮DETCs的圆,ImageJ软件的使用。仅拍摄 DETC(γδTCR 阳性细胞)的图像 > 使用 ImageJ 上的多边形选择工具为每个 DETC 标记轮廓 > 分析 >设置测量 > 区域和形状描述(选择“形状描述符”参数进行测量和显示)> 分析> 测量或 Ctrl+M。这将显示一个带有测量值的表格。对WT 和 SnTg 皮肤部分中的所有DETC执行此步骤。圆度值为 1 表示单元格是一个完美的圆,接近 0 的值表示单元格被拉长。在此协议中,我们使用了≥ 0.5的阈值循环值来调用 DETC 活动。使用这些测量值绘制的图(图 5)显示K14 Snail 转基因皮肤中的 DETC更圆,表明与 WT 皮肤中的相比,DETC具有激活作用。
 
 
图 5. 在 DETC 中观察到的形态变化的量化。激活DETCs显示形态变化小号从一个树枝状到圆形形状,这是使用量化的ImageJ软件。散点图显示,与 WT 表皮中的 DETC 相比,Snail 转基因表皮中的 DETC 更圆(圆度值 ≥ 0.5)。数据代表值 ± SEM,*** P < 0.0001,基于学生t检验。
 
食谱
 
P BS用2 ×抗生素
将 2 ml青霉素-链霉素(100 × ) 添加到 10 ml 10 × PBS 中,并用 ddH 2 O使体积达到 100 ml 。
无钙E培养基
如Nowak 和 Fuchs ( 2009)所述,为表皮角质形成细胞制备不含钙的 E 培养基。
FACS 染色缓冲液
2毫升螯合胎牛血清
1 ml 青霉素-链霉素 (100 × )
100 微升 50 毫克/毫升庆大霉素
添加到 5 ml 的 10 × PBS 中
B环体积高达 50 ml,使用 ddH 2 O
RPMI 媒体
10% 胎牛血清
2 mM 谷氨酰胺
500 µl 青霉素-链霉素 (100 × )
50 μ中号β巯基乙醇
25 mM HEPES
1 mM 丙酮酸钠
100 µM 非必需氨基酸
20 U/ml重组人IL-2
B环体积高达 500 ml,使用 RPMI 培养基
0.2% 海卫 X-100
加入2mL 100%的Triton X - 100到100毫升10 × PBS,并把体积达到1升用的DDH 2 ö 。
用于透化的封闭缓冲液
加0.5 ml山羊血清(或血清源自相同物种作为次级荧光缀合的抗体)和10 μ升的Triton X -在1毫升10的100 × PBS和使体积至10ml的DDH 2 O.
4% 多聚甲醛 (PFA)
通过将 12.5 ml 16% PFA 添加到 5 ml 10 × PBS 中稀释 10 × PBS 中的 16% 多聚甲醛,并通过添加ddH 2 O使体积达到 50 ml 。
70% 乙醇
通过在 300毫升蒸馏水中加入 700 毫升 100% 乙醇,在蒸馏水中稀释 100% 乙醇。
莫维尔
将 2.4 克 MOWIOL ® 4-88添加到4.75 毫升甘油中并搅拌混合。
加入 6 ml 水并在室温下搅拌数小时。
加入 12 ml 0.2 M Tris (pH 8.5) 并加热至 50°C 10 分钟,偶尔混合。
MOWIOL 4-88 溶解后,以 5 , 000 × g离心15 分钟澄清溶液。
 
致谢
 
作者要感谢 Binita Dam 和 Johan Ajnabi 对手稿的批判性审查。Jamora实验室的工作得到了 inStem 核心基金和印度政府生物技术部过去的资助 (BT/PR8738/AGR/36/770/2013);国立关节炎、肌肉骨骼和皮肤病研究所 (NIAMS),NIH (5R01AR053185-03);和美国癌症协会 (15457-RSG-08-164-01-DDC)。动物工作得到了生物技术系国家小鼠研究资源 (NaMoR) (BT/PR5981/MED/31/181/2012; 2013-2016) 的部分支持。我们感谢 NCBS/inStem 动物护理和资源中心的工作人员对畜牧业的帮助,以及 NCBS/inStem 中央成像和流式细胞仪 (CIFF) 的工作人员在图像采集方面的帮助。我们致力于此协议内存的我们的同事和朋友温迪哈夫兰教授谁是在该领域的先驱和领导γδ T细胞生物学。
该协议用于以下手稿:Lee, P., Gund, R., Dutta, A., Pincha, N., Rana, I., Ghosh, S., Witherden, D., Kandyba, E., MacLeod , A., Kobielak, K., Havran, WL 和 Jamora, C. (2017)。 通过 IL-1 依赖性激活γδ T 细胞来刺激毛囊干细胞增殖。Elife 6 :e28875 。
 
利益争夺
 
没有需要申报的利益冲突。
 
伦理
 
所有与动物有关的工作都是在 NCBS/inStem 动物护理和资源中心进行的,并且协议得到了机构动物伦理委员会的批准。
 
参考
 
Badarinath, K.、Dutta, A.、Hegde, A.、Pincha, N.、Gund, R. 和 Jamora, C.(2019 年)。表皮角质形成细胞、树突状表皮 T 细胞和毛囊干细胞之间的相互作用。分子生物学方法1879:285-297。
Boismenu, R. 和 Havran, WL (1994)。上皮内γδ T 细胞对上皮细胞生长的调节。 科学266(5188):1253-1255。
Cruz, MS, Diamond, A., Russell, A. 和 Jameson, JM (2018)。皮肤免疫和疾病中的人类αβ和γδ T 细胞。前免疫学9:1304。
De Craene, B.、Denecker, G.、Vermassen, P.、Taminau, J.、Mauch, C.、Derore, A.、Jonkers, J.、Fuchs, E. 和 Berx, G. (2014)。表皮 Snail 表达通过增强细胞保护、表皮干/祖细胞扩增和增强转移潜能来驱动皮肤癌的发生和进展。细胞死亡差异21(2):310-320。
Du, F., Nakamura, Y., Tan, TL, Lee, P., Lee, R., Yu, B. 和 Jamora, C. (2010)。蜗牛在表皮角质形成细胞中的表达促进有利于肿瘤形成的皮肤炎症和增生。癌症研究70(24):10080-10089。
Edelbaum, D., Mohamadzadeh, M., Bergstresser, PR, Sugamura, K. 和 Takashima, A. (1995)。白细胞介素(IL)-15促进鼠表皮生长γδ通过涉及第一个机构T细胞ë β -和γ ç的IL-2受体的-chains。J Invest Dermatol 105(6): 837-843。             
Fischer, AH、Jacobson, KA、Rose, J. 和 Zeller, R. (2008)。冷冻切片组织。CSH 协议2008:pdb prot4991。              
Gay, D., Kwon, O., Zhang, Z., Spata, M., Plikus, MV, Holler, PD, Ito, M., Yang, Z., Treffeisen, E., Kim, CD, Nace, A ., Zhang, X., Baratono, S., Wang, F., Ornitz, DM, Millar, SE 和 Cotsarelis, G. (2013)。来自真皮γδT细胞的Fgf9在受伤后诱导毛囊新生。国家医学19(7): 916-923。              
Gund, R.、Zirmire, R.、J, H.、Kansagara, G. 和 Jamora, C.(2021 年)。受伤皮肤中干细胞增殖和再上皮化的组织学和免疫组织化学检查。生物协议11(2): e3894。
Gustafsson, K.、Herrmann, T. 和 Dieli, F.(2020 年)。社论:了解γδ T 细胞多功能性 - 走向免疫治疗应用。 前免疫学11: 921。
Havran, WL, Grell, S., Duwe, G., Kimura, J., Wilson, A., Kruisbeek, AM, O'Brien, RL, Born, W., Tigelaar, RE 和 Allison, JP (1989)。V γ 3 特异性单克隆抗体揭示的鼠 Thy-1+ 树突状表皮细胞的 T 细胞受体 γ 链表达的多样性有限。Proc Natl Acad Sci USA 86(11): 4185-4189。
夏皮罗,H 。M. (2003)。实用流式细胞术。约翰威利父子公司
Jameson, J. 和 Havran, WL (2007)。皮肤γδ T 细胞在体内平衡和伤口愈合中发挥作用。 免疫学修订版215:114-122。              
Jameson, J.、Ugarte, K.、Chen, N.、Yachi, P.、Fuchs, E.、Boismenu, R. 和 Havran, WL (2002)。皮肤γδ T 细胞在伤口修复中的作用。科学296(5568):747-749。
Kashem, SW 和 Kaplan, DH (2018)。通过酶消化分离鼠皮肤常驻和迁移树突细胞。Curr Protoc Immunol 121(1): e45。
李,P 。,李,D 。Ĵ 。,陈,C 。,陈,S 。w ^ 。,陈,我。和贾莫拉,C 。(2009)表皮半胱天冬酶 8 的动态表达模拟伤口愈合反应。自然458(7237):519-23。
Lee, P., Gund, R., Dutta, A., Pincha, N., Rana, I., Ghosh, S., Witherden, D., Kandyba, E., MacLeod, A., Kobielak, K., Havran, WL 和 Jamora, C. (2017)。通过 IL-1 依赖性激活γδ T 细胞刺激毛囊干细胞增殖。Elife 6 :e28875。
Li, F.、Adase, CA 和 Zhang, LJ (2017)。新生和成年小鼠皮肤原代小鼠角质形成细胞的分离和培养。J Vis Exp (125) : 56027。
Nielsen, MM, Lovato, P., MacLeod, AS, Witherden, DA, Skov, L., Dyring-Andersen, B., Dabelsteen, S., Woetmann, A., Odum, N., Havran, WL, Geisler, C. 和 Bonefeld, CM (2014)。接触超敏反应中树突状表皮 T 细胞的 IL-1beta 依赖性激活。J Immunol 192(7): 2975-2983。              
Nowak, JA 和 Fuchs, E. (2009)。上皮干细胞的分离和培养。方法 Mol Biol 482:215-232。              
Sharp, LL、Jameson, JM、Cauvi, G. 和 Havran, WL (2005)。树突状表皮 T 细胞通过局部产生胰岛素样生长因子 1 来调节皮肤稳态。Nat Immunol 6(1): 73-79。              
Wohn, CT, Pantelyushin, S., Ober-Blobaum, JL 和 Clausen, BE (2014)。Aldara 诱导的银屑病样皮肤炎症:皮肤树突细胞和先天淋巴细胞的分离和表征。分子生物学方法1193:171-185。
 

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Copyright Rana et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rana, I., Badarinath, K., Zirmire, R. K. and Jamora, C. (2021). Isolation and Quantification of Mouse γδT-cells in vitro and in vivo. Bio-protocol 11(17): e4148. DOI: 10.21769/BioProtoc.4148.
  2. Lee, P., Gund, R., Dutta, A., Pincha, N., Rana, I., Ghosh, S., Witherden, D., Kandyba, E., MacLeod, A., Kobielak, K., Havran, W. L. and Jamora, C. (2017). Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells. Elife 6: e28875.
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