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Aug 2018

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Ultrasound Guided Intra-thymic Injection to Track Recent Thymic Emigrants and Investigate T Cell Development
超声引导胸腺内注射并追踪新近胸腺迁出细胞和T细胞发育观测   

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Abstract

To track recent thymic emigrants (RTEs) or study T cell development in the thymus, intra-thymic injection of a cellular tag or precursor cells for various T cell lineages is often desired. However, the traditional surgical approach to expose the thymus for intra-thymic injection is time-consuming and can cause a high level of pain and stress to animals, which might disrupt immune homeostasis, potentially confounding the results. Here, we introduce an ultrasound guided intra-thymic injection procedure, which is non-surgical and minimally invasive to animals. This technique is relatively easy to learn and offers an efficient and accurate tool to track RTEs or perform intra-thymic transfer of various cell types to investigate their differentiation.

Keywords: Ultrasound (超声), Intra-thymic injection (胸腺内注射), Recent thymic emigrants (新近胸腺迁出细胞), Thymus (胸腺), T cell development (T细胞发育), Invariant natural killer T (iNKT) cells (恒定型自然杀伤T细胞), Regulatory T (Treg) cells (调节性T细胞)

Background

T cells play an essential role in protective immunity against invading pathogens and malignant self. The thymus is the site where T cells originate and develop; therefore, it has long been an organ of interest to study immune system development. Many of these studies involve tracking recent thymic emigrants (RTEs) or dissecting the differentiation steps of T cells into different lineages. The labeling of thymocytes via intra-thymic injection of a cellular tag like a biotinylating agent (NHS-biotin) or Fluorescein isothiocyanate (FITC), allows specific detection of RTEs in the periphery. Furthermore, it is often necessary to perform direct intra-thymic transfer of precursor cells to investigate precursor-product relationships during T cell development. As such, the ultrasound guided intra-thymic injection procedure described here provides a fast, easily learned and non-invasive technique to study such processes in the thymus.

The traditional intra-thymic injection approach involves surgical procedures that cut the sternum to open the thoracic cavity to directly visualize and inject the thymus. Though this method has been used extensively, this technique is invasive and subjects animals to stress and possible mortality. Also, performing this surgical intra-thymic injection requires ample experience in mouse surgery. Furthermore, the efficiency and accuracy of this technique sometimes can be questionable, as the visualization of thymus is usually limited. To improve the efficiency, accuracy and success rate of intra-thymic injection, and reduce the stress of animals during the procedure, we describe here a non-surgical, minimally invasive ultrasound guided intra-thymic injection procedure. Using ultrasound imaging, the thymus can be clearly visualized without any surgical exposure, and accurate injection into the thymus can be further guided and monitored. We have successfully implemented this ultrasound guided intra-thymic injection approach in a variety of studies. As shown here and the original research papers (Wang and Hogquist, 2018; Owen et al., 2018), we performed intra-thymic injection of biotin to label thymocytes and tracked the biotin+ RTEs in the periphery. Moreover, we were able to identify and sort precursor cells for invariant natural killer T (iNKT) cells and regulatory T (Treg) cells which, upon intra-thymic transfer into congenic hosts, substantially differentiated into all three iNKT effector subsets or mature Treg cells, respectively. Lastly, we also transferred purified wild-type (Wt) DN thymocytes into congenic recipients using ultrasound guided intra-thymic injection and detected robust development into CD4+ CD8+ double positive (DP), CD4+ single positive and CD8+ single positive thymocytes. Such a technique could be combined with genetic manipulation of DN thymocytes in order to identify cell-intrinsic factors important for T cell differentiation. In general, the ultrasound guided intra-thymic injection could serve as a convenient tool for further investigation of T cell emigration and development in the murine thymus.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (DOT Scientific, catalog number: 509-FTG)
  2. 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097)
  3. 70 μm cell strainers (Corning, Falcon®, catalog number: 352350)
  4. 6-well plates (Corning, Costar®, catalog number: 3506)
  5. 0.5 ml insulin syringes (EXELINT INTERNATIONAL, catalog number: 26028)
  6. 3 ml syringes (Covidien, catalog number: 8881513918)
  7. 5 ml FACS tubes with Cell-Strainer Cap (Corning, Falcon®, catalog number: 352235)
  8. 96 round-bottom well plates (SARSTEDT, catalog number: 82.1582.001)
  9. Aluminum foil (Spring Grove)
  10. MACS LS columns (Miltenyi Biotec, catalog number: 130-042-401)
  11. Facemask (3M, catalog number: 1820)
  12. Medical tape (3M Transpore, catalog number: 1527-1; 3M Durapore, catalog number: 1538-1)
  13. Paper towel
  14. Alcohol pad (McKESSON, catalog number: 58-204)
  15. Cotton pad (McKESSON, catalog number: 44082000)
  16. B6 (C57BL/6NCr) mice (THE JACKSON LABORATORY, catalog number: 000664)
  17. B6.SJL (B6-LY5.2/Cr) mice (THE JACKSON LABORATORY, catalog number: 002014)
  18. Tbx21GFP KN2 BALB/cBYJ mice (have been previously described in Wang and Hogquist, 2018)
  19. Foxp3GFP (B6.Cg-Foxp3tm2(EGFP)Tch/J) mice (THE JACKSON LABORATORY, catalog number: 006772)
  20. CD45.1+ BALB/cBYJ (CByJ.SJL(B6)-Ptprca/J) (THE JACKSON LABORATORY, catalog number: 006584)
  21. Eye ointment (MAJOR LubriFresh P.M., catalog number: 0904-6488-38)
  22. Nair (NairTM HAIR REMOVER LOTION)
  23. Ultrasound gel (aquasonic CLEAR®, catalog number: 03-08)
  24. Phosphate buffered saline (PBS) (Corning, Mediatech, catalog number: 21-040-CV)
  25. Isoflurane (Piramal Healthcare, catalog number: 001725CS)
  26. Anti-Biotin MicroBeads (Miltenyi Biotec, catalog number: 130-105-637)
  27. Viability dye Ghost DyeTM Red 780 (TONBO Biosciences, catalog number: 13-0865-T100)
  28. CD1d-tetramer (PBS57-loaded CD1d biotinylated monomers were from NIH tetramer core)
  29. Streptavidin-PE (BD Biosciences, catalog number: 554061)
  30. Streptavidin-BV421 (BioLegend, catalog number: 405225)
  31. Anti-CCR7 (BD Biosciences, catalog number: 562675)
  32. Anti-CD4 (BD Biosciences, catalog number: 563331)
  33. Anti-CD8α (BD Biosciences, catalog number: 563786)
  34. Anti-CD24 (BioLegend, catalog number: 101824)
  35. Anti-NK1.1 (BioLegend, catalog number: 108718)
  36. Anti-CD44 (TONBO Biosciences, catalog number: 80-0441-U025)
  37. Anti-human CD2 (BioLegend, catalog number: 309218)
  38. Anti-TCRβ (BD Biosciences, catalog number: 563221)
  39. Anti-CD45.1 (BioLegend, catalog number: 110738)
  40. Anti-CD45.2 (eBioscience, catalog number: 11-0454-81)
  41. Anti-PLZF (BD Biosciences, catalog number: 563490)
  42. Anti-ROR-γt (BD Biosciences, catalog number: 562684)
  43. Anti-T-bet (BioLegend, catalog number: 644824)
  44. Anti-CD25 (TONBO Biosciences, catalog number:65-0251-U100)
  45. Anti-CD45.1 (BD Biosciences, catalog number: 563754)
  46. Anti-CD90.2 (eBioscience, catalog number: 47-0902-82)
  47. Anti-CD90.1 (eBioscience, catalog number: 57-0900-82)
  48. Anti-CD73 (eBioscience, catalog number: 48-0731-82)
  49. Sulfo-NHS-LC biotin (Thermo Fisher Scientific, catalog number: 21335)
  50. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150), heat inactivated at 65 °C
  51. Ethylenediaminetetraacetate acid (EDTA) (Fisher Scientific, catalog number: BP120-500)
  52. Sodium azide (Fisher Scientific, catalog number: BP922I-500)
  53. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514-500G)
  54. Potassium bicarbonate (KHCO3) (Fisher Scientific, catalog number: P235-500)
  55. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906)
  56. autoMACS Rinsing Solution (Miltenyi Biotec, catalog number: 130-091-222)
  57. MACS BSA Stock Solution (Miltenyi Biotec, catalog number: 130-091-376)
  58. Betadine (BETADINE Surgical Scrub, catalog number: 67618-151-17)
  59. FACS buffer (see Recipes)
  60. ACK lysis buffer (see Recipes)
  61. MACS buffer (see Recipes)

Equipment

  1. Class-II biosafety cabinet/laminar flow hood
  2. Vevo® 2100 Imaging System (Visual Sonics, see Figure 1A)
  3. Acrylic chamber (see Figure 1B)
  4. MACS Multistand (Miltenyi Biotec, catalog number: 130-042-303)
  5. QuadroMACS Separator (Miltenyi Biotec, catalog number: 130-090-976)
  6. Benchtop centrifuge (Beckman Coulter, model: Allegra X-12-R)
  7. Hemacytometer (Sigma-Aldrich, catalog number: Z359629-1EA)
  8. BD Fortessa H0081 Flow cytometer
  9. BD FACSAria II Cell Sorter
  10. MS550 transducer (Visual Sonics, see Figure 1A)
  11. Refrigerator (2-8 °C; Isotemp LABORATORY REFRIGIRATOR)
  12. Isoflurane vaporizer
  13. Heating pad

Software

  1. FlowJo 10.4.0 (https://www.flowjo.com/)

Procedure

  1. Ultrasound guided intra-thymic injection
    1. Pick up one mouse and place in the acrylic chamber connected to an isoflurane vaporizer (Figures 1A and 1B). We normally use the mouse ranges from 6 to 10 weeks of age. 
    2. Induce anesthesia using the acrylic chamber with 3% of isoflurane in O2 administered at a rate of 600 ml/min into the chamber (Figure 1B).
    3. Frequently check the anesthesia state of the mouse; the mouse should be anesthetized within 3 min.
    4. Take mouse out of the chamber and quickly transfer to the immobilization heating pad, cover the mouse nose with a facemask to maintain anesthesia with 3% of isoflurane in O2 administered at a rate of 600 ml/min (Figure 1C).
    5. Apply eye ointment on both eyes to prevent them from drying during anesthesia period.
    6. Flip the mouse belly up and immobilize limbs with medical tape, during which the mouse should be maintained under anesthesia with the facemask (Figure 1C).
    7. Apply Nair on chest area, wait for 1-2 min to effect, remove hair with a paper towel. Swipe the chest area with betadine and clean up with an alcohol pad to sterilize the area.
    8. Apply ultrasound gel on the chest area and then perform the ultrasound scan using the Vevo® 2100 Imaging System (Visual Sonics) with the MS550 transducer, which ranges from 32 to 40 MHz. Fix the transducer right above the chest area in direct contact with ultrasound gel; the live ultrasound imaging should be as shown, while the syringe will approach the thymus from the side of chest (Figures 1C and 1D).
    9. Identify the thymus in the ultrasound image as shown in Figure 1D.
    10. Fill the syringe (29G1/2 needle) with 10-20 µl of NHS-biotin at 1 mg/ml in PBS or with cell suspension, tightly fix the syringe in the syringe stand on the right side of the animal and adjust to approximate 135° angle point from the ground (Figure 1C).
    11. First, approach the needle into the gel between mouse and transducer until clearly visualized in the ultrasound imaging as a high-density white dot (Figure 1E). Then, slide the syringe up and down to target the needle directly above the thymus (Figure 1E).
    12. Pull the needle out of the gel, lower the syringe (till the needle tip points to the chest area), advance the needle into the mouse through the ribs and continuously penetrate toward the thymus. Frequently monitor the ultrasound image until the needle tip is visualized within the area of thymus (Figure 1F), perform injection, then pull the needle out of the mouse.
    13. After injection, remove the ultrasound gel using a cotton pad and wipe the chest area clean with an alcohol pad. Remove medical tape restraints on mouse limbs, remove mouse from nose cone and place the mouse back in the cage for recovery. The mouse should recover in 2-3 min and behave similar to before injection.


      Figure 1. Ultrasound imaging system and ultrasound guided intra-thymic injection. A. Vevo® 2100 Imaging System for ultrasound imaging of mouse, including the acrylic chamber and the immobilization heating pad. B. Mouse in the acrylic chamber for anesthesia. C. Mouse immobilized on the heating pad for ultrasound imaging and intra-thymic injection. D. The ultrasound imaging of mouse chest area before injection, the white dashed line outlines the thymus. E. The white dashed line outlines the thymus; the yellow arrow indicates the needle tip in the gel right above the thymus. F. The white dashed line outlines the thymus; the yellow arrow indicates the needle penetrating inside the thymus during injection. G. Image for the localization of thymus in the chest cavity of mouse. The yellow arrow indicates the thymus.

  2. Track recent thymic emigrants (RTEs) in the periphery
    1. To track RTEs, perform intra-thymic injection of NHS-biotin as described in Procedure A. 
    2. Euthanize the mouse 24-72 h later, collect the thymus (Figure 1G) in ice cold FACS buffer and spleen in ice cold MACS buffer.
    3. Prepare single cell suspension from the thymus and spleen by mashing the tissues with the plunger of a 3 ml syringe and passing cell suspension through a 70 μm cell strainer.
    4. Determine the cell count of each sample using hemacytometer.
    5. In accordance with the Miltenyi protocol, resuspend spleen cells in 80 μl of MACS buffer per 107 cells, add 20 μl of Anti-Biotin MicroBeads per 107 cells. Vortex to mix well and incubate for 45 min in the refrigerator (2-8 °C).
    6. Wash cells with 2 ml of MACS buffer per 107 cells and centrifuge to pellet at 300 x g for 10 min. Aspirate supernatant.
    7. After wash, resuspend cell pellet with 500 μl of MACS buffer up to 108 cells.
    8. Proceed to the magnetic enrichment of biotin+ cells using LS columns according to the Miltenyi protocol.
    9. Collect the flowthrough and bound fraction of cells and perform flow cytometry to identify biotin+ cells through staining with streptavidin-PE or streptavidin-BV421.

  3. Developmental outcome of transferred progenitors
    1. Perform intra-thymic injection of sorted or purified cells of choice into congenically distinct recipient mice as described in Procedure A to study T cell development in the thymus. In our hands, we successfully transferred invariant natural killer T (iNKT) progenitor cells, T regulatory (Treg) progenitor cells and DN thymocytes, and monitored their differentiation into iNKT effector subsets, mature Treg cells, and mature SP thymocytes, respectively. 
    2. Briefly, sort the iNKT progenitors from thymocytes of T-betGFP/KN2 mice, Treg progenitors from thymocytes of Foxp3GFP mice with a FACS sorter, or purify and enrich the DN thymocytes using LS column by depleting CD4+, CD8+ and CD4+ CD8+ thymocytes of B6 mice. More details concerning the background of iNKT cell and Treg cell development, as well as gating and sorting strategies of their precursors were shown in the original research paper (Wang and Hogquist, 2018; Owen et al., 2018).
    3. Wash the sorted or purified cells 2 times with 10 ml of PBS and centrifuge to pellet at 300 x g for 10 min. Aspirate supernatant.
    4. After the second wash, resuspend cell pellet in about 20 μl of PBS, keep cell suspension on ice before injection.
    5. Perform the intra-thymic injection of the cell suspension as described in Procedure A.
    6. Euthanize the mouse after the injection (experimental end points should be determined empirically for each study), collect the thymus in ice cold FACS buffer.
    7. Prepare single cell suspension from the thymus by mashing the tissues with the plunger of a 3 ml syringe and passing cell suspension through a 70 μm cell strainer.
    8. Perform flow cytometry to identify the transferred cells through staining of the congenic markers (CD45.1 and CD45.2) and/or other related cell markers.

Data analysis

  1. The analysis of flow cytometry data was performed using Flowjo 10.
  2. Intra-thymic injection of NHS-biotin provided robust labeling of nearly half of the total thymocytes (Figure 2A), more results were shown in Figure 1–Figure supplement 3 of the original research paper (Wang and Hogquist, 2018).
  3. To track the RTEs in the periphery, mice were euthanized, and spleens were collected for analysis 24 h after intra-thymic injection of biotin. RTEs were identified as biotin+ cells in the periphery. Since RTEs were rare events in the peripheral T cells, magnetic enrichment of biotin+ cells was performed (see Procedure B for detailed protocol), and this approach provided clear detection of biotin+ cells to track RTEs (Figure 2B). More results were shown in Figure 1–Figure supplement 3 and Figures 2A-2B of the original research paper (Wang and Hogquist, 2018).


    Figure 2. Ultrasound guided intra-thymic injection of biotin to track RTEs. A. Representative flow cytometry plots of biotinylated cells revealed by streptavidin-BV421 staining in total thymocytes 24 h later after intra-thymic injection of PBS (left column) or NHS-biotin (right column). B. Twenty-four hours after the intra-thymic injection of NHS-biotin, biotin+ RTEs could be specifically identified in the spleen CD4+ (upper row) or CD8+ (bottom row) T cells populations using magnetic enrichment. C. Frequency of biotin+ cells revealed by streptavidin-PE/BV421 staining in CD4 SP, CD8 SP and total thymocytes in the thymus 24 h after intra-thymic injection of NHS-biotin.

  4. iNKT progenitors were intra-thymically transferred into congenic distinct recipients, and 5 days later, a substantial differentiation into all three iNKT effector subsets was detected. More results were shown in Figures 1E and 1F of original research paper (Wang and Hogquist, 2018).
  5. Immature Treg progenitors were intra-thymically transferred into congenic distinct recipients, and 6 days later, significant differentiation into CD25+ Foxp3+ mature Tregs was detected. More results were shown in Figure 1 of original research paper (Owen et al., 2018).
  6. DN thymocytes were purified and enriched using LS column (Figure 3A) and were intra-thymically transferred into congenic distinct recipients (Figure 3B). The recipient mice were irradiated sub-lethally (500 Rads) the day before receiving intra-thymic injection. 14 days later, the transferred cells could be readily identified as CD45.1+ cells, and a substantial differentiation into DP thymocytes, CD4+, and CD8+ thymocytes was detected (Figure 3B).


    Figure 3. Intra-thymic transfer of DN thymocytes for investigation of T cells development. A. CD4 and CD8 expression before (left column) and after (right column) the purification of DN thymocytes. B. The purified CD45.1+ DN thymocytes or PBS were intra-thymically transferred in CD45.2+ congenic recipients and transferred cells could be specifically detected according to their expression of CD45.1 and CD45.2 (left two columns); 14 days after intra-thymic transfer, the DN thymocytes showed robust development into DP thymocytes, CD4+ and CD8+ thymocytes (right bottom row) similar to the endogenous thymocytes (right upper row).

Recipes

  1. FACS buffer (store at 4 °C, ~10 ml for each sample)
    2% FBS
    0.02% sodium azide
    500 ml PBS
  2. ACK lysis buffer (store at 4 °C, ~5 ml for each sample)
    150 mM NH4Cl
    10 mM KHCO3
    0.1 mM EDTA
    1 L ddH2O
    Adjust pH to 7.2-7.4
  3. MACS buffer (store at 4 °C, ~30 ml for each sample)
    1,450 ml autoMACS Rinsing Solution
    75 ml MACS BSA Stock Solution

Acknowledgments

This work was supported by R37AI39560 (KAH), R01AI124512 (MAF), UMN doctoral dissertation fellowship (HW), Immunology training grant 2T32AI007313 (DLO).

Competing interests

The authors declare no competing financial interests.

Ethics

All animal experimentation in this protocol was approved by the University of Minnesota Institutional Animal Care and Use Committee.

References

  1. Wang, H. and Hogquist, K. A. (2018). CCR7 defines a precursor for murine iNKT cells in thymus and periphery. Elife 7: e34793.
  2. Owen, D. L., Mahmud, S. A., Sjaastad, L. E., Williams, J. B., Spanier, J. A., Simeonov, D. R., Ruscher, R., Huang, W., Proekt, I., Miller, C. N., Hekim, C., Jeschke, J. C., Aggarwal, P., Broeckel, U., LaRue, R. S., Henzler, C. M., Alegre, M., Anderson, M. S., August, A., Marson, A., Zheng, Y., Williams, C. B., and Farrar, M. A. (2018). Thymic regulatory T cells arise via two distinct developmental programs. (Unpublished)

简介

为了追踪最近的胸腺移植物(RTE)或研究胸腺中的T细胞发育,通常需要胸腺内注射用于各种T细胞谱系的细胞标签或前体细胞。 然而,用于暴露胸腺以进行胸腺内注射的传统手术方法是耗时的并且可以对动物造成高水平的疼痛和压力,这可能破坏免疫稳态,可能使结果混淆。 在这里,我们介绍了超声引导胸内注射程序,这是非手术和动物微创。 该技术相对容易学习,并提供有效且准确的工具来跟踪RTE或进行各种细胞类型的胸腺内转移以研究它们的分化。

【背景】T细胞在抵抗入侵病原体和恶性自身的保护性免疫中起重要作用。胸腺是T细胞起源和发展的部位;因此,长期以来它一直是研究免疫系统发育的一个感兴趣的器官。这些研究中的许多涉及追踪最近的胸腺移民(RTEs)或将T细胞的分化步骤解剖成不同的谱系。通过胸腺内注射细胞标签如生物素化剂(NHS-生物素)或异硫氰酸荧光素(FITC)标记胸腺细胞允许特异性检测外周的RTE。此外,经常需要进行前体细胞的直接胸腺内转移以研究T细胞发育期间的前体 - 产物关系。因此,这里描述的超声引导的胸腺内注射程序提供了一种快速,易于学习和非侵入性的技术来研究胸腺中的这种过程。

传统的胸腺内注射方法涉及切开胸骨以打开胸腔以直接观察和注射胸腺的外科手术。虽然这种方法已被广泛使用,但这种技术具有侵入性,使动物受到压力和可能的死亡。此外,进行这种手术胸内注射需要在小鼠手术中有丰富的经验。此外,这种技术的效率和准确性有时可能是有问题的,因为胸腺的可视化通常是有限的。为了提高胸内注射的效率,准确性和成功率,并减少手术过程中动物的压力,我们在此描述了非手术,微创超声引导胸内注射手术。使用超声成像,胸腺可以在没有任何手术暴露的情况下清晰可见,并且可以进一步引导和监测对胸腺的精确注射。我们在各种研究中成功实施了这种超声引导胸内注射方法。如本文所示和原始研究论文(Wang和Hogquist,2018; Owen et al。>,2018),我们进行胸腺内注射生物素以标记胸腺细胞并追踪生物素 + < / sup>外围的RTE。此外,我们能够识别和分选不变的自然杀伤T(iNKT)细胞和调节性T(Treg)细胞的前体细胞,这些细胞在胸腺内转移到同类宿主中时,基本上分化为所有三种iNKT效应子亚群或成熟Treg细胞。 , 分别。最后,我们还使用超声引导的胸腺内注射将纯化的野生型(Wt)DN胸腺细胞转移到同基因受体中,并检测到CD4 + CD8 + 双阳性的强烈发展( DP),CD4 + 单阳性和CD8 + 单阳性胸腺细胞。这种技术可以与DN胸腺细胞的遗传操作相结合,以鉴定对T细胞分化重要的细胞内在因子。一般而言,超声引导的胸腺内注射可以作为进一步研究小鼠胸腺中T细胞迁移和发育的便利工具。

关键字:超声, 胸腺内注射, 新近胸腺迁出细胞, 胸腺, T细胞发育, 恒定型自然杀伤T细胞, 调节性T细胞

材料和试剂

  1. 1.5 ml微量离心管(DOT Scientific,目录号:509-FTG)
  2. 15毫升锥形离心管(Corning,Falcon ®,目录号:352097)
  3. 70μm细胞过滤器(Corning,Falcon ®,目录号:352350)
  4. 6孔板(Corning,Costar ®,目录号:3506)
  5. 0.5 ml胰岛素注射器(EXELINT INTERNATIONAL,目录号:26028)
  6. 3毫升注射器(Covidien,目录号:8881513918)
  7. 带有Cell-Strainer Cap的5 ml FACS管(Corning,Falcon ®,目录号:352235)
  8. 96个圆底孔板(SARSTEDT,目录号:82.1582.001)
  9. 铝箔(Spring Grove)
  10. MACS LS柱(Miltenyi Biotec,目录号:130-042-401)
  11. 面罩(3M,目录号:1820)
  12. 医用胶带(3M Transpore,目录号:1527-1; 3M Durapore,目录号:1538-1)
  13. 纸巾
  14. 酒精垫(McKESSON,目录号:58-204)
  15. 棉垫(McKESSON,目录号:44082000)
  16. B6(C57BL / 6NCr)小鼠(THE JACKSON LABORATORY,目录号:000664)
  17. B6.SJL(B6-LY5.2 / Cr)小鼠(THE JACKSON LABORATORY,目录号:002014)
  18. Tbx21 > GFP KN2 BALB / cBYJ小鼠(先前已在Wang和Hogquist中描述,2018)
  19. Foxp3 > GFP (B6.Cg-Foxp3tm2(EGFP)Tch / J)小鼠(THE JACKSON LABORATORY,目录号:006772)
  20. CD45.1 + BALB / cBYJ(CByJ.SJL(B6)-Ptprca / J)(THE JACKSON LABORATORY,目录号:006584)
  21. 眼膏(MAJOR LubriFresh P.M.,目录号:0904-6488-38)
  22. Nair(Nair TM HAIR REMOVER LOTION)
  23. 超声波凝胶(aquasonic CLEAR ®,目录号:03-08)
  24. 磷酸盐缓冲盐水(PBS)(Corning,Mediatech,目录号:21-040-CV)
  25. 异氟醚(Piramal Healthcare,目录号:001725CS)
  26. 抗生物素MicroBeads(Miltenyi Biotec,目录号:130-105-637)
  27. 活力染料Ghost Dye TM Red 780(TONBO Biosciences,目录号:13-0865-T100)
  28. CD1d-四聚体(载有PBS57的CD1d生物素化单体来自NIH四聚体核心)
  29. Streptavidin-PE(BD Biosciences,目录号:554061)
  30. Streptavidin-BV421(BioLegend,目录号:405225)
  31. 抗CCR7(BD Biosciences,目录号:562675)
  32. 抗CD4(BD Biosciences,目录号:563331)
  33. 抗CD8α(BD Biosciences,目录号:563786)
  34. Anti-CD24(BioLegend,目录号:101824)
  35. Anti-NK1.1(BioLegend,目录号:108718)
  36. 抗CD44(TONBO Biosciences,目录号:80-0441-U025)
  37. 抗人类CD2(BioLegend,目录号:309218)
  38. 抗TCRβ(BD Biosciences,目录号:563221)
  39. Anti-CD45.1(BioLegend,目录号:110738)
  40. Anti-CD45.2(eBioscience,目录号:11-0454-81)
  41. 抗PLZF(BD Biosciences,目录号:563490)
  42. 抗ROR-γt(BD Biosciences,目录号:562684)
  43. Anti-T-bet(BioLegend,目录号:644824)
  44. 抗CD25(TONBO Biosciences,目录号:65-0251-U100)
  45. 抗CD45.1(BD Biosciences,目录号:563754)
  46. Anti-CD90.2(eBioscience,目录号:47-0902-82)
  47. Anti-CD90.1(eBioscience,目录编号:57-0900-82)
  48. Anti-CD73(eBioscience,目录号:48-0731-82)
  49. Sulfo-NHS-LC生物素(Thermo Fisher Scientific,目录号:21335)
  50. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150),在65°C下加热灭活
  51. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:BP120-500)
  52. 叠氮化钠(Fisher Scientific,目录号:BP922I-500)
  53. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A4514-500G)
  54. 碳酸氢钾(KHCO 3 )(Fisher Scientific,目录号:P235-500)
  55. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A7906)
  56. autoMACS Rinsing Solution(Miltenyi Biotec,目录号:130-091-222)
  57. MACS BSA Stock Solution(Miltenyi Biotec,目录号:130-091-376)
  58. Betadine(BETADINE Surgical Scrub,目录号:67618-151-17)
  59. FACS缓冲液(见食谱)
  60. ACK裂解缓冲液(参见食谱)
  61. MACS缓冲区(见食谱)

设备

  1. II级生物安全柜/层流罩
  2. Vevo ® 2100成像系统(Visual Sonics,见图1A)
  3. 亚克力室(见图1B)
  4. MACS Multistand(Miltenyi Biotec,目录号:130-042-303)
  5. QuadroMACS分离器(Miltenyi Biotec,目录号:130-090-976)
  6. 台式离心机(Beckman Coulter,型号:Allegra X-12-R)
  7. 血细胞计数器(Sigma-Aldrich,目录号:Z359629-1EA)
  8. BD Fortessa H0081流式细胞仪
  9. BD FACSAria II细胞分选仪
  10. MS550换能器(Visual Sonics,见图1A)
  11. 冰箱(2-8°C; Isotemp LABORATORY REFRIGIRATOR)
  12. 异氟醚蒸发器
  13. 加热垫

软件

  1. FlowJo 10.4.0( https://www.flowjo.com/ )

程序

  1. 超声引导胸内注射
    1. 拿起一只小鼠并放置在与异氟烷蒸发器连接的丙烯酸室中(图1A和1B)。我们通常使用6到10周龄的小鼠。&nbsp;
    2. 使用丙烯酸室诱导麻醉,其中O 2 中的3%异氟烷以600ml / min的速率进入腔室(图1B)。
    3. 经常检查小鼠的麻醉状态;小鼠应在3分钟内麻醉。
    4. 将小鼠从室中取出并快速转移至固定加热垫,用面罩覆盖小鼠鼻子以维持麻醉,用O 2 中的3%异氟烷以600ml / min的速率给药(图1C)。
    5. 在双眼上涂抹眼膏,以防止它们在麻醉期间干燥。
    6. 翻转鼠标腹部并用医用胶带固定肢体,在此期间应使用面罩将小鼠保持在麻醉下(图1C)。
    7. 在胸部涂抹Nair,等待1-2分钟,用纸巾擦去头发。用聚维酮碘皂擦拭胸部区域并用酒精垫清洁以对该区域进行消毒。
    8. 在胸部区域涂抹超声凝胶,然后使用Vevo ® 2100成像系统(Visual Sonics)和MS550传感器进行超声波扫描,范围为32至40 MHz。将换能器固定在胸部区域正上方,与超声凝胶直接接触;实时超声成像应如图所示,而注射器将从胸腔侧接近胸腺(图1C和1D)。
    9. 识别超声图像中的胸腺,如图1D所示。
    10. 在注射器(29G1 / 2针)中加入10-20μl含1 mg / ml的NHS生物素的PBS或细胞悬液,将注射器紧紧固定在动物右侧的注射器支架上并调节至约135 °角度离地面(图1C)。
    11. 首先,将针接近小鼠和换能器之间的凝胶,直到在超声成像中清晰可见为高密度白点(图1E)。然后,上下滑动注射器,将针头直接定位在胸腺上方(图1E)。
    12. 将针头从凝胶中拉出,放下注射器(直到针尖指向胸部区域),通过肋骨将针头推入鼠标并持续穿透胸腺。经常监测超声图像直到针尖在胸腺区域内可见(图1F),进行注射,然后将针头拉出鼠标。
    13. 注射后,用化妆棉取下超声波凝胶,并用酒精垫擦拭胸部区域。取下鼠标肢体上的医用胶带束缚,从鼻锥上取下鼠标,将鼠标放回笼子中进行恢复。小鼠应在2-3分钟内恢复,并且表现与注射前相似。


      图1.超声成像系统和超声引导胸内注射。 A. Vevo ® 2100成像系统用于小鼠的超声成像,包括丙烯酸室和固定加热垫。 B.小鼠在丙烯酸室中进行麻醉。 C.固定在加热垫上的小鼠,用于超声成像和胸腺内注射。 D.注射前小鼠胸部区域的超声成像,白色虚线勾勒出胸腺。 E.白色虚线勾勒出胸腺;黄色箭头表示胸腺正上方凝胶中的针尖。 F.白色虚线勾勒出胸腺;黄色箭头表示针在注射过程中穿透胸腺内部。 G.胸腺在胸腔内定位的图像。黄色箭头表示胸腺。

  2. 追踪周边最近的胸腺移民(RTEs)
    1. 要跟踪RTE,按照程序A中的描述进行胸腺内注射NHS-生物素。&nbsp;
    2. 24-72小时后对小鼠实施安乐死,在冰冷的FACS缓冲液中收集胸腺(图1G),在冰冷的MACS缓冲液中收集脾脏。
    3. 通过用3ml注射器的柱塞捣碎组织并使细胞悬浮液通过70μm细胞过滤器,从胸腺和脾中制备单细胞悬液。
    4. 使用血细胞计数器确定每个样品的细胞计数。
    5. 根据Miltenyi方案,将脾细胞重悬于80μlMACS缓冲液/ 10×7×/ ml细胞中,每10 7 细胞加入20μl抗生物素微珠。涡旋混匀,在冰箱(2-8℃)中孵育45分钟。
    6. 每10个细胞用2ml MACS缓冲液洗涤细胞,离心,以300×10g /小时沉淀10分钟。吸出上清液。
    7. 洗涤后,用500μlMACS缓冲液重悬细胞沉淀至10 8 细胞。
    8. 根据Miltenyi方案,使用LS柱进行生物素 + 细胞的磁性富集。
    9. 收集细胞的流穿和结合部分并进行流式细胞术以通过用链霉抗生物素蛋白-PE或链霉抗生物素蛋白-BV421染色来鉴定生物素 + 细胞。

  3. 转移祖细胞的发育结果
    1. 如方法A中所述,将选择的分选或纯化的细胞胸腺内注射到先天性不同的受体小鼠中以研究胸腺中的T细胞发育。在我们的手中,我们成功转移了不变的自然杀伤T(iNKT)祖细胞,T调节(Treg)祖细胞和DN胸腺细胞,并分别监测它们分化为iNKT效应子集,成熟Treg细胞和成熟SP胸腺细胞。&nbsp;
    2. 简而言之,用FACS分选仪从T-betGFP / KN2小鼠的胸腺细胞,来自 Foxp3 > GFP 小鼠的胸腺细胞的Treg祖细胞中分选iNKT祖细胞,或纯化和富集DN胸腺细胞使用LS柱消除B6小鼠的CD4 + ,CD8 + 和CD4 + CD8 + 胸腺细胞。关于iNKT细胞和Treg细胞发育的背景以及它们前体的门控和分选策略的更多细节在原始研究论文中显示(Wang和Hogquist,2018; Owen et al。>,2018 )。
    3. 用10ml PBS洗涤分选或纯化的细胞2次,并离心以300μL/小时沉淀10分钟。吸出上清液。
    4. 在第二次洗涤后,将细胞沉淀重悬于约20μlPBS中,在注射前将细胞悬浮液保持在冰上。
    5. 如程序A所述,进行胸腺内注射细胞悬液。
    6. 注射后使小鼠安乐死(实验终点应根据经验确定每个研究),在冰冷的FACS缓冲液中收集胸腺。
    7. 通过用3ml注射器的柱塞捣碎组织并使细胞悬浮液通过70μm细胞过滤器,从胸腺制备单细胞悬浮液。
    8. 进行流式细胞术以通过染色同基因标记物(CD45.1和CD45.2)和/或其他相关细胞标记物来鉴定转移的细胞。

数据分析

  1. 使用Flowjo 10进行流式细胞术数据的分析。
  2. 胸腺内注射NHS-生物素为近一半的胸腺细胞提供了强有力的标记(图2A),更多的结果显示在原始研究论文的图1-Figure补充3中(Wang和Hogquist,2018)。
  3. 为了追踪外围的RTE,对小鼠实施安乐死,并在胸腺内注射生物素后24小时收集脾脏用于分析。 RTE被鉴定为外周的生物素 + 细胞。由于RTE是外周T细胞中的罕见事件,因此进行了生物素 + 细胞的磁性富集(详见方案参见程序B),该方法提供了生物素 + 细胞跟踪RTE(图2B)。更多结果显示在原始研究论文(Wang和Hogquist,2018)的图1 - 图补充3和图2A-2B中。


    图2.超声引导胸内注射生物素以追踪RTE。 A.在胸腺内注射PBS后24小时,通过链霉抗生物素蛋白-BV421染色显示生物素化细胞的代表性流式细胞仪图(左栏)或NHS-生物素(右栏)。 B.胸腺内注射NHS-生物素后24小时,可在脾CD4 + (上排)或CD8中特异性鉴定生物素 + RTEs sup> + (底行)使用磁性富集的T细胞群。 C.胸腺内注射NHS-生物素后24小时胸腺中CD4 SP,CD8 SP和胸腺总胸腺中链霉抗生物素蛋白-PE / BV421染色显示生物素 + 细胞的频率。

  4. 将iNKT祖细胞在胸腺内转移到同基因不同的受体中,并且在5天后,检测到实质上分化成所有三种iNKT效应子集。更多结果显示在原始研究论文的图1E和1F中(Wang和Hogquist,2018)。
  5. 将未成熟的Treg祖细胞在胸腺内转移到同基因的不同受体中,并且6天后,检测到显着分化成CD25 + Foxp3 + 成熟的Treg。更多结果显示在原始研究论文的图1中(Owen et al。>,2018)。
  6. 使用LS柱纯化和富集DN胸腺细胞(图3A),并将胸腺内转移到同基因不同的受体中(图3B)。在接受胸腺内注射前一天,对受体小鼠进行亚致死剂量(500 Rads)照射。 14天后,转移的细胞可以很容易地鉴定为CD45.1 + 细胞,并且显着分化为DP胸腺细胞,CD4 + 和CD8 + < / sup>检测到胸腺细胞(图3B)。


    图3. DN胸腺细胞的胸腺内转移用于研究T细胞发育。 :一种。 CD4和CD8表达之前(左列)和之后(右列)DN胸腺细胞的纯化。 B.纯化的CD45.1 + DN胸腺细胞或PBS在CD45.2 + 同类受体中进行胸腺内转移,转移细胞可根据其表达特异性检测。 CD45.1和CD45.2(左两列);胸腺内转移后14天,DN胸腺细胞显示出与胸腺细胞,CD4 + 和CD8 + 胸腺细胞(右下排)相似的内源性胸腺细胞(右侧)上排)。

食谱

  1. FACS缓冲液(储存在4°C,每个样品约10 ml)
    2%FBS
    0.02%叠氮化钠
    500毫升PBS
  2. ACK裂解缓冲液(储存在4°C,每个样品约5 ml)
    150mM NH 4 Cl
    10mM KHCO 3
    0.1 mM EDTA
    1 L ddH 2 O
    将pH调节至7.2-7.4
  3. MACS缓冲液(4°C储存,每个样品约30 ml)
    1,450毫升autoMACS冲洗液
    75毫升MACS BSA库存解决方案

致谢

该工作得到R37AI39560(KAH),R01AI124512(MAF),UMN博士论文奖学金(HW),免疫学培训基金2T32AI007313(DLO)的支持。

利益争夺

作者声明没有竞争性的经济利益。

伦理

该方案中的所有动物实验均由明尼苏达大学机构动物护理和使用委员会批准。

参考

  1. Wang,H。和Hogquist,K。A.(2018)。 CCR7定义了胸腺和外周的小鼠iNKT细胞的前体。 Elife > 7:e34793。
  2. Owen,DL,Mahmud,SA,Sjaastad,LE,Williams,JB,Spanier,JA,Simeonov,DR,Ruscher,R.,Huang,W.,Proekt,I.,Miller,CN,Hekim,C.,Jeschke, JC,Aggarwal,P.,Broeckel,U.,LaRue,RS,Henzler,CM,Alegre,M.,Anderson,MS,August,A.,Marson,A.,Zheng,Y.,Williams,CB,and Farrar ,MA(2018)。胸腺调节性T细胞通过两种不同的发育程序产生。 (未公开)
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Wang 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. Wang, H., Owen, D. L., Qian, L. J., Chopp, L. B., Farrar, M. A. and Hogquist, K. A. (2018). Ultrasound Guided Intra-thymic Injection to Track Recent Thymic Emigrants and Investigate T Cell Development. Bio-protocol 8(23): e3107. DOI: 10.21769/BioProtoc.3107.
  2. Wang, H. and Hogquist, K. A. (2018). CCR7 defines a precursor for murine iNKT cells in thymus and periphery. Elife 7: e34793.
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