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Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs
小鼠肺CD4+ T细胞的鼻内/血管内抗体同时标记

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Abstract

CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in different anatomic locations in mouse lungs.

Keywords: Memory CD4+ T cell(记忆CD4+ T细胞), Lung(肺), Bronchoalveolar lavage fluid(支气管肺泡灌洗液), Intranasal/intravascular antibody labeling(鼻内/血管内抗体标记), Flow cytometry(流式细胞术)

Background

To distinguish memory T cells in the circulation and tissues, a method for intravascular staining of T cells has been developed (Anderson et al., 2012). This method has been widely used to define memory T cells in many organs and tissues, including lungs, spleens and intestines. However, memory T cells in the respiratory tract are located in three unique anatomic locations, i.e., airway, parenchyma and pulmonary vasculature. Intravascular staining cannot distinguish cells in the airway and parenchyma, since they are both isolated from the circulation and intravascularly-administered antibodies will not stain these two populations. We designed a simultaneous intranasal/intravascular antibody labeling assay that can label and distinguish cells in all three locations using minimal amount of antibodies.

Materials and Reagents

  1. 1.5 ml microtubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69715 )
  2. 1 ml insulin syringe fitted with 28 G x ½ needle (BD, catalog number: 329420 )
  3. Precision glide needles (20 G x 1) (BD, catalog number: 305175 )
  4. 5 ml polystyrene round bottom tubes (Corning, Falcon®, catalog number: 352054 )
  5. Precision glide needles (25 G x 5/8) (BD, catalog number: 305122 )
  6. 200 μl tips (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12655 )
  7. 15 ml screw cap conical tubes (SARSTEDT, catalog number: 62.554.002 )
  8. 10 ml syringes (BD, catalog number: 309604 )
  9. 12 well cell culture plates (Corning, Costar®, catalog number: 3513 )
  10. 3 ml syringes (BD, catalog number: 309657 )
  11. 50 ml screw cap conical tubes (SARSTEDT, catalog number: 62.547.004 )
  12. Cell strainers (70 µm nylon) (Corning, Falcon®, catalog number: 352350 )
  13. 60 x 15 mm tissue culture dish (Corning, Costar®, catalog number: 353802 )
  14. 10 ml stripettes (Corning, Costar®, catalog number: 3548 )
  15. Gauze sponges (4 x 4 inch) (Pro Advantage by NDC, catalog number: P157118 )
  16. 0.2 μm filter (EMD Millipore, catalog numbers: SCGPU11RE and SLGP033RS )
  17. 2.5 ml graduate transfer pipettes (RPI, catalog number: 147501-1S )
  18. Absorbent pads (COVIDIEN, catalog number: 949 )
  19. Mouse
  20. CD45-brilliant violet 510 (BV510) (Clone: 30-F11) (BioLegend, catalog number: 103138 )
  21. 1x Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  22. CD90.2-APC-eFluor 780 (Clone: 53-2.1) (Affymetrix, eBioscience, catalog number: 47-0902 )
  23. Isoflurane (USP inhalation vapour, liquid) (Drugs, catalog number: 57319-559-06 )
  24. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  25. Trypan blue solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  26. CD16/32-PerCP/Cy5.5 (Clone: 93) (BioLegend, catalog number: 101324 )
  27. CD4-FITC (Clone: RM4-5) (BioLegend, catalog number: 100510 )
  28. 2,2,2-tribromoethanol (Sigma-Aldrich, catalog number: T48402 )
  29. 2-methyl-2-butanol (Sigma-Aldrich, catalog number: 152463 )
  30. Nano-pure water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  31. RPMI medium 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  32. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  33. L-glutamine (200 mM) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  34. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  35. MEM non-essential amino acids solution 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  36. Sodium pyruvate 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  37. Penicillin/streptomycin 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  38. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  39. Collagenase D (Roche Diagnostics, catalog number: 11088882001 )
  40. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  41. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025 )
  42. Sodium azide, NaN3 (AMRESCO, catalog number: 0639 )
  43. Potassium bicarbonate, KHCO3 (Sigma-Aldrich, catalog number: 237205 )
  44. Ammonium chloride, NH4Cl (Sigma-Aldrich, catalog number: A9434 )
  45. EDTA-Na2 (Sigma-Aldrich, catalog number: E9884 )
  46. BD Cytofix fixation solution (BD, catalog number: 554655 )
  47. Avertin (see Recipes)
  48. Complete RPMI 1640 medium (see Recipes)
  49. Digestion buffer (see Recipes)
  50. FACS buffer (see Recipes)
  51. ACK lysis buffer (see Recipes)

Equipment

  1. Desiccator (SP Scienceware - Bel-Art Products - H-B Instrument, model: Space Saver Vacuum Desiccators )
  2. Fume hood (LABSCAPE)
  3. Pipetman P10 (Eppendorf, model: Research plus )
  4. Pipetman P200 (Eppendorf, model: Research plus )
  5. Pipetman P1000 (Eppendorf, model: Research plus )
  6. Pipet aid (Eppendorf, model: Eppendorf Easypet 3 )
  7. Heat lamp (Whitehead Industrial, catalog number: 30715 )
  8. Mouse restrainer (Braintree Scientific, catalog number: TV-150 STD )
  9. Spray bottle with 70% ethanol (SKS Science Products, catalog number: 0185-11 )
  10. Surgical scissors (Sklar Surgical Instrument, catalog number: 47-1246 )
  11. Tweezers (Sklar Surgical Instrument, catalog number: 66-7644 )
  12. Polystyrene foam (Regular polystyrene box top)
  13. Rocker (Labnet, catalog number: S0500 )
  14. Refrigerated tabletop centrifuge (Beckman Coulter, model: Allegra 6R )
  15. Hemocytometer (Thermo Fisher Scientific, catalog number: 99503 )
  16. Flow cytometer (BD, model: FACSVerse)

Software

  1. Flowjo software (Version 10.0.7)

Procedure

  1. Simultaneous intranasal/intravascular antibody labeling (see Videos 1 and 2)
    1. Dilute 0.25 μg CD45-BV510 antibody in 100 μl DPBS in a 1.5 ml microtube (for intranasal labelling). Dilute 0.5 μg CD90.2-APC-eFuor 780 antibody in 300 μl DPBS in another 1.5 ml microtube and load the antibody into a 1 ml insulin syringe with a 28 G x ½ needle (for intravenous labelling). Place the microtube and the syringe on ice until use. (see Note 1)
    2. Put a plastic desiccator in a fume hood. Place some gauze in the bottom chamber followed by 5 ml of isoflurane. (see Note 2)
    3. Place a mouse on the grate inside the desiccator (see Note 3). 
    4. Load a P200 micropipette with 100 µl of CD45-BV510 antibody.
    5. Immediately after the rate of respiration slows, pick up the mouse, hold it vertically and slowly deliver 100 µl of CD45-BV510 antibody onto the mouse nostrils. Hold the mouse upright so that the liquid on the nostrils is completely aspirated into the lungs.
    6. Place the mouse back in the cage. After 2 min, turn on the heat lamp over the cage and warm up the mouse for 1 min.
    7. Remove the mouse from the cage, put it in a mouse restrainer and inject 0.5 μg CD90.2-APC-eFuor 780 antibody in 300 μl DPBS through the tail vein with the 1 ml insulin syringe prepared in point 1.
    8. Release the mouse from the restrainer and immediately anesthetize the mouse by intraperitoneal injection of 300 µl of avertin solution. Secure the mouse in the palm of the hand and hold it horizontally, then insert the needle at a shallow angle tangential to the mouse and slowly dispense avertin solution into the peritoneal cavity.

      Video 1. Intranasal administration of antibodies

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      Video 2. Intravenous administration of antibodies

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  2. Harvest cells from airway (see Video 3)

    Video 3. Harvest of bronchoalveolar fluid, to obtain airway resident cells

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    1. Prepare a 1 ml syringe with 20 G x 1 needle. Carefully pass about 1.25 inches 20 G tubing onto the needle. Sharpen the end of the tubing with scissors. Load the syringe with 1 ml complete RPMI 1640 medium and put it on ice until use (Figure 1).


      Figure 1. Representative pictures of syringe preparation and surgery for BALF lavage

    2. When the mouse is fully anesthetized, typically within 2 min, immobilize it on a piece of polystyrene foam with absorbent pad on top by pinning each limb with a 25 G x 5/8 needle.
    3. Use a spray bottle filled with 70% ethanol to wet the fur of the mouse.
    4. Cut open the skin covering the abdominal and thoracic cavities through to the lower jaw. Use scissors to remove the tissue from the neck and expose the trachea.
    5. Use tweezers to separate the trachea from the underlying tissue and place a 200 μl tip under the trachea.
    6. Use a razor to cut through ½ of the trachea. Insert the 1 ml syringe with 20 G x 1 needle into the incision. Lavage for 4 times with complete RPMI 1640 medium. Collect the bronchoalveolar lavage fluid (BALF) in a 15 ml conical tube on ice. Typically 3-3.5 ml of BALF is recovered (Figure 1).
    7. Spin down cells for 5 min at 400 x g at 4 °C in a bucket tabletop centrifuge and resuspend the cells in 1 ml of ice cold FACS buffer (see Note 5).

  3. Sacrifice the mouse and harvest cells from lung (see Video 4)

    Video 4. Perfusing and harvesting lungs for cellular analysis

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    1. After collection of BALF, cutting open the abdominal cavity to expose the underside of the diaphragm. Cut through the diaphragm with scissors and then remove the ribcage to fully expose the heart and lung.
    2. Fill a 10 ml syringe with ice cold sterile DPBS and attach a 25 G x 5/8 needle. Insert the needle into the left ventricle of the heart and smoothly dispense 5 ml of DPBS into the heart. In the meantime, use tweezers to break the right atria to allow blood to drain from circulation. Remove the needle from the left ventricle and insert it into the right ventricle to directly perfuse the lung with the remaining 5 ml of DPBS. (see Note 6)
    3. Cut the heart away from the lung and then remove the lung from the thoracic cavity after cutting the trachea and any remaining connective tissue.
    4. Place the lung into the well of a 12 well tissue culture plate filled with 2.5 ml of DPBS on ice.
    5. Rinse the lung with DPBS and transfer it into another well without DPBS. Mince the lungs into very fine pieces using scissors.
    6. Transfer minced lung with a 2.5 ml transfer pipette to 5 ml of digestion buffer in a 15 ml conical tube.
    7. Place tubes on a rocker and gently rotate at room temperature for 30 min in the dark.
    8. Place a 70 µm cell strainer into a 60 x 15 mm tissue culture dish.
    9. Transfer lung tissue in digestion buffer to the cell strainer using a 2.5 ml transfer pipette. Gently press and dissociate tissue through strainer with the flat end of a 3 ml syringe plunger. Process tissues until there is only connective tissue remaining on the strainer and rinse the strainer with complete RPMI 1640 medium. Transfer the resulting suspension to a 50 ml conical tube.
    10. Spin down lung cells for 5 min at 400 x g at 4 °C in a bucket tabletop centrifuge.
    11. Pour off supernatant and resuspend the cells in 3 ml of ACK buffer for 1 min to lyse the remaining red blood cells. Neutralize the ACK buffer with 30 ml of ice cold DPBS.
    12. Spin down the cells for 5 min at 400 x g at 4 °C and resuspend the cells in 5 ml of ice cold FACS buffer.

  4. Cell staining
    1. Count the cells from BALF and lung using a hemocytometer in the presence of trypan blue.
    2. Spin down lung cells for 5 min at 400 x g at 4 °C and resuspend the cells in FACS buffer at 1 million cells per 50 µl.
    3. Spin down cells in the BALF for 5 min at 400 x g at 4 °C. Since much fewer cells are recovered from airway, resuspend 1-5 x 105 cells per 50 μl FACS buffer as needed.
    4. Dilute 0.25 μg CD4-FITC and 0.1 μg CD16/32 antibodies in 50 μl FACS buffer (see Note 4). 
    5. Gently mix 50 μl cells and 50 μl antibodies together in a FACS tube.
    6. Incubate the cells in the dark for 15 min at 4 °C.
    7. Wash the cells once with 2 ml of FACS buffer at 400 x g for 5 min at 4 °C.
    8. Remove the supernatant and resuspend the cells in 100 µl FACS buffer.
    9. Pass the cells through a 70 µm cell strainer into new FACS tubes using a pipetman. Acquire FACS data using a flow cytometer and analyze data using Flowjo software (Figure 2). (see Note 7)
    10. Flow cytometry gating strategy


      Figure 2. Gating strategy to distinguish CD4+ T cells in the airway, parenchyma and pulmonary vasculature. To localize memory CD4+ T cells in the respiratory tract, 0.25 μg of fluorochrome-conjugated CD45 and 0.5 μg of fluorochrome-conjugated CD90.2 antibody were injected into a SARS-CoV nulceocapsid protein-experienced mouse by i.n. and i.v. routes, respectively. Cells in the airway and lung were then harvested, stained and collected as described in Experimental Procedures and analysis using Flowjo software. A. CD4+ T cells in the airway: CD4+CD45+CD90.2-. B. CD4+ T cells in the parenchyma: CD4+CD45-CD90.2-. CD4+ T cells in the vasculature: CD4+CD45-CD90.2+. Data are representative of 10 independent experiments. (Zhao et al., 2016)

Notes

  1. CD45 and CD90 are expressed by all CD4+ T cells. Both CD45 and CD90 antibodies can be used to stain CD4+ T cells in the lung and circulation. Phycoerythrin (PE), APC, BV (or their derivatives)-conjugated antibodies are preferentially chosen since these molecules have large molecular weights that will minimize airway/vascular leakage during in vivo labeling.
  2. Make sure to tape over the center hole in the plastic desiccator grate because small mice are sometimes able to squeeze through this hole and jump into the bottom, isoflurane-filled chamber.
  3. Here we used an antigen-experienced mouse that had been immunized with a construct expressing the SARS-CoV nucleocapsid protein (Zhao et al., 2016). This protocol can be applied to study memory and effector as well as naïve CD4+ T cells in mouse lungs.
  4. Cells can also be stained with other phenotypic (surface) antibodies or cultured for intracellular cytokine staining as previously described (Fett et al., 2014).
  5. For phenotypic staining, cells will be resuspended in FACS buffer. For intracellular staining, cell will be resupended in complete RPMI 1640 medium.
  6. The exterior surface of the lung will turn white after a good perfusion. Generally 5 ml PBS is sufficient.
  7. This step is necessary to prevent clots formation in the flow cytometer. Other flow cytometers, such as LSR, Fortessa… can also be used to acquire cells.

Recipes

  1. Avertin (250 ml)
    5 g 2,2,2-tribromoethanol
    5 ml 2-methyl-2-butanol
    225 ml hot nano-pure water (95 °C)
    Stir for 2 h in a fume hood and adjust volume to 250 ml
    0.2 μm filter, aliquot and store at -20 °C
  2. Complete RPMI 1640 medium (500 ml)
    417 ml RPMI 1640 medium
    12.5 ml 1 M HEPES
    5 ml 100x L-glutamine
    50 ml FBS
    5 ml 100x MEM non-essential amino acids
    5 ml 100x sodium pyruvate
    5 ml 100x penicillin/streptomycin
    0.5 ml 2-mercaptoethanol (50 mM in nano-pure H2O, 0.2 μm filtered)
    Store at 4 °C
  3. Digestion buffer (100 ml)
    100 mg collagenase D
    10 mg DNase I
    2 ml FBS
    1 ml 100x glutamine
    2.5 ml HEPES (1 M)
    1 ml 100x penicillin/streptomycin
    Adjust volume to 100 ml with HBSS
    Aliquot and store at -20 °C
  4. FACS buffer (500 ml)
    1.7 ml sodium azide (from 30% stock made in nano-pure H2O)
    15 ml FBS
    483.3 ml DPBS
    Store at 4 °C
  5. ACK lysis buffer
    1 g KHCO3
    8.3 g NH4Cl
    37.2 mg EDTA-Na2
    Adjust volume to 1,000 ml with nano-pure H2O and filter through a 0.2 μm filter
    Store at room temperature

Acknowledgments

This work was supported by the Thousand Talents Plan Award of China 2015 (J.Z.), the Municipal Healthcare Joint-Innovation Major Project of Guangzhou (201604020011) (J.Z.) and the N.I.H. (USA) (PO1 AI060699, S.P.). The protocol described herein was based on the following manuscript: Zhao et al. Immunity. 2016 Jun 21; 44(6):1379-91.

References

  1. Anderson, K. G., Sung, H., Skon, C. N., Lefrancois, L., Deisinger, A., Vezys, V. and Masopust, D. (2012). Cutting edge: intravascular staining redefines lung CD8 T cell responses. J Immunol 189(6): 2702-2706.
  2. Fett, C., Zhao, J. and Perlman, S. (2014). Measurement of CD8 and CD4 T cell responses in mouse lungs. Bio Protoc 4(6).
  3. Zhao, J., Zhao, J., Mangalam, A. K., Channappanavar, R., Fett, C., Meyerholz, D. K., Agnihothram, S., Baric, R. S., David, C. S. and Perlman, S. (2016). Airway memory CD4+ T cells mediate protective immunity against emerging respiratory coronaviruses. Immunity 44(6): 1379-1391.

简介

许多呼吸道病毒感染中CD4细胞反应已被证明是保护性的。在呼吸道中,CD4 + T细胞包括气道中的细胞和附着于肺血管的细胞和细胞。在这里,我们详细讨论了可用于表征小鼠肺不同解剖位置的CD4 + T细胞的方法。

背景 为了区分循环和组织中的记忆T细胞,已经开发了一种T细胞血管内染色的方法(Anderson等人,2012)。这种方法被广泛用于定义许多器官和组织(包括肺,脾和肠)的记忆T细胞。然而,呼吸道中的记忆T细胞位于三个独特的解剖位置,即气道,薄壁组织和肺血管。血管内染色不能区分气道和实质中的细胞,因为它们都是从循环中分离的,血管内施用的抗体不会污染这两个群体。我们设计了同时进行的鼻内/血管内抗体标记测定,可以使用最少量的抗体来标记和区分所有三个位置的细胞。

关键字:记忆CD4+ T细胞, 肺, 支气管肺泡灌洗液, 鼻内/血管内抗体标记, 流式细胞术

材料和试剂

  1. 1.5ml微管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:69715)
  2. 装有28 G x½针的1 ml胰岛素注射器(BD,目录号:329420)
  3. 精密滑动针(20 G x 1)(BD,目录号:305175)
  4. 5 ml聚苯乙烯圆底管(Corning,Falcon ®,目录号:352054)
  5. 精密滑动针(25 G x 5/8)(BD,目录号:305122)
  6. 200μl提示(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12655)
  7. 15毫升螺旋锥形管(SARSTEDT,目录号:62.554.002)
  8. 10ml注射器(BD,目录号:309604)
  9. 12孔细胞培养板(Corning,Costar ®,目录号:3513)
  10. 3 ml注射器(BD,目录号:309657)
  11. 50毫升螺旋锥形管(SARSTEDT,目录号:62.547.004)
  12. 细胞过滤器(70μm尼龙)(Corning,Falcon ®,目录号:352350)
  13. 60×15mm组织培养皿(Corning,Costar ®,目录号:353802)
  14. 10毫升剥皮板(Corning,Costar ®,目录号:3548)
  15. 纱布海绵(4 x 4英寸)(Pro Advantage,NDC,目录号:P157118)
  16. 0.2μm过滤器(EMD Millipore,目录号:SCGPU11RE和SLGP033RS)
  17. 2.5 ml毕业转移移液器(RPI,目录号:147501-1S)
  18. 吸收垫(COVIDIEN,目录号:949)
  19. 鼠标
  20. CD45-亮紫510(BV510)(克隆:30-F11)(BioLegend,目录号:103138)
  21. 1x Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190-144)
  22. CD90.2-APC-eFluor 780(克隆:53-2.1)(Affymetrix,eBioscience,目录号:47-0902)
  23. 异氟烷(USP吸入蒸气,液体)(药品,目录号:57319-559-06)
  24. 乙醇(Sigma-Aldrich,目录号:459836)
  25. 台盼蓝溶液(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  26. CD16/32-PerCP/Cy5.5(克隆:93)(BioLegend,目录号:101324)
  27. CD4-FITC(克隆:RM4-5)(BioLegend,目录号:100510)
  28. 2,2,2-三溴乙醇(Sigma-Aldrich,目录号:T48402)
  29. 2-甲基-2-丁醇(Sigma-Aldrich,目录号:152463)
  30. 纳米纯水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  31. RPMI培养基1640(Thermo Fisher Scientific,Gibco TM,目录号:11875093)
  32. HEPES(1 M)(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  33. L-谷氨酰胺(200mM)100x(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  34. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150)
  35. MEM非必需氨基酸溶液100x(Thermo Fisher Scientific,Gibco TM,目录号:11140050)
  36. 丙酮酸钠100x(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  37. 青霉素/链霉素100x(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  38. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  39. 胶原酶D(Roche Diagnostics,目录号:11088882001)
  40. DNase I(Roche Diagnostics,目录号:10104159001)
  41. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025)
  42. 叠氮化钠,NaN 3(AMRESCO,目录号:0639)
  43. 碳酸氢钾,KHCO 3(Sigma-Aldrich,目录号:237205)
  44. 氯化铵,NH 4 Cl(Sigma-Aldrich,目录号:A9434)
  45. EDTA-Na 2(Sigma-Aldrich,目录号:E9884)
  46. BD Cytofix固定液(BD,目录号:554655)
  47. Avertin(见食谱)
  48. 完成RPMI 1640培养基(见食谱)
  49. 消化缓冲液(参见食谱)
  50. FACS缓冲区(见配方)
  51. ACK裂解缓冲液(参见食谱)

设备

  1. 干燥器(SP科学软件 - 贝尔艺术产品 - H-B仪器,型号:节省空间的真空干燥器)
  2. 通风柜(LABSCAPE)
  3. Pipetman P10(Eppendorf,型号:Research plus)
  4. Pipetman P200(Eppendorf,model:Research plus)
  5. Pipetman P1000(Eppendorf,型号:Research plus)
  6. 吸管辅助(Eppendorf,型号:Eppendorf Easypet 3)
  7. 加热灯(Whitehead Industrial,目录号:30715)
  8. 鼠标限制器(Braintree Scientific,目录号:TV-150 STD)
  9. 用70%乙醇喷雾瓶(SKS Science Products,目录号:0185-11)
  10. 手术剪刀(Sklar Surgical Instrument,目录号:47-1246)
  11. 镊子(Sklar Surgical Instrument,目录号:66-7644)
  12. 聚苯乙烯泡沫(普通聚苯乙烯盒顶)
  13. 摇杆(Labnet,目录号:S0500)
  14. 冷藏台式离心机(Beckman Coulter,型号:Allegra 6R)
  15. 血细胞计数器(Thermo Fisher Scientific,目录号:99503)
  16. 流式细胞仪(BD,型号:FACSVerse)

软件

  1. Flowjo软件(10.0.7版)

程序

  1. 同时鼻内/血管内抗体标记(见视频1和2)
    1. 稀释0.25μgCD45-BV510抗体在100μlDPBS 1.5ml微管(用于鼻内标记)。将稀释0.5μgCD90.2-APC-eFor 780抗体在另一个1.5ml微量管中的300μlDPBS中,并将抗体加载到具有28G×½针(用于静脉内标记)的1ml胰岛素注射器中。将微管和注射器放在冰上,直到使用。 (见注1)
    2. 将塑料干燥器放在通风橱中。将一些纱布放在底部腔室中,然后加入5ml异氟烷。 (见注2)
    3. 将鼠标放在干燥器内的格栅上(见注3)。 
    4. 加载带有100μlCD45-BV510抗体的P200微量移液器。
    5. 在呼吸速度慢后,拿起小鼠,将其垂直握住并缓慢地将100μl的CD45-BV510抗体递送到小鼠鼻孔上。按住鼠标直立,使鼻孔上的液体完全吸入肺部。
    6. 将鼠标放回笼子里。 2分钟后,打开笼罩上的加热灯,并将鼠标预热1分钟。
    7. 将小鼠从笼中取出,将其放入小鼠限制器中,并通过尾静脉注射0.5μgCD90.2-APC-eForor 780抗体,在300μlDPBS中,用1ml在第1点制备的胰岛素注射器。
    8. 从限制器释放鼠标,并通过腹膜内注射300μl的黄芩苷溶液立即麻醉小鼠。将鼠标固定在手掌中并水平握住,然后将针头插入与鼠标相切的浅角度,并慢慢地将Avertin溶液分配到腹膜腔中。

      <! - flashid2099v154开始 - >
      视频1.抗体的鼻内给药
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      视频2.抗体的静脉内施用
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  2. 从气道收获细胞(见视频3)

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    视频3.支气管肺泡液收获,获得呼吸道居民细胞
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    1. 准备1 g注射器,20 G x 1针。小心地将大约1.25英寸的20 G管道传递到针上。用剪刀剪开管道的末端。用1ml完整RPMI 1640培养基装入注射器,放在冰上直至使用(图1)

      图1. BALF灌洗注射器制备和手术的代表性图片

    2. 当鼠标完全麻醉时,通常在2分钟内,通过用25 G x 5/8针钉扎每个肢体将其固定在一块聚苯乙烯泡沫上,吸收垫在顶部。
    3. 使用装有70%乙醇的喷雾瓶润湿鼠标毛。
    4. 切开覆盖腹腔和胸腔的皮肤通过下颌。用剪刀从颈部取出组织,暴露气管。
    5. 使用镊子将气管与底层组织分开,并在气管下方放置200μl提示。
    6. 使用剃刀切开气管的1/2。将1 ml注射器用20 G x 1针插入切口。用完整的RPMI 1640培养基灌洗4次。在冰上将15ml锥形管收集支气管肺泡灌洗液(BALF)。通常3-3.5ml的BALF被回收(图1)
    7. 在四台桌面离心机中,在4℃下以400×x 将细胞旋转5分钟,并将细胞重新悬浮在1ml冰冷的FACS缓冲液中(见注5)。

  3. 牺牲小鼠并从肺部收获细胞(见视频4)

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    视频4.进行细胞分析的灌注和收获肺
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    1. 收集BALF后,切开腹腔露出隔膜下侧。用剪刀穿过隔膜,然后取出胸腔以彻底暴露心肺。
    2. 在冰冷的无菌DPBS上注入10ml注射器,并附上25 G x 5/8针。将针插入心脏的左心室,并顺利地将5ml DPBS注入心脏。同时,使用镊子打破右心房,让血液从流通中排出。从左心室取出针头并将其插入右心室,直接用剩余的5ml DPBS灌注肺。 (见注6)
    3. 切开心脏远离肺部,然后切开气管和剩余的结缔组织后,从胸腔取出肺。
    4. 将肺置于装有2.5ml DPBS的12孔组织培养板的孔中。
    5. 用DPBS冲洗肺并将其转移到没有DPBS的另一口中。用剪刀将肺部变成非常细的碎片。
    6. 将切碎的肺用2.5ml转移移液管转移到5ml锥形管中的5ml消化缓冲液中
    7. 将管放在摇杆上,并在室温下轻轻地在黑暗中旋转30分钟。
    8. 将70μm的细胞过滤器放入60 x 15 mm的组织培养皿中
    9. 将消化缓冲液中的肺组织使用2.5ml转移移液管转移至细胞过滤器。用3 ml注射器柱塞的平端轻轻按压和离开组织通过过滤器。过滤组织,直到只有结缔组织残留在过滤器上,并用完整的RPMI 1640培养基冲洗过滤器。将得到的悬浮液转移到50ml锥形管中
    10. 在四层桌面离心机中,在4℃下以400 x x x x x x x x x X x x x x x x x x x x x x x x x x x x x x x x x x x x x x
    11. 倒出上清液并将细胞重悬于3ml的ACK缓冲液中1分钟以裂解剩余的红细胞。用30毫升冰冷的DPBS中和ACK缓冲区。
    12. 在4℃下以400×g将细胞旋转细胞5分钟,并将细胞重悬于5ml冰冷的FACS缓冲液中。

  4. 细胞染色
    1. 在台盼蓝存在下使用血细胞计数器从BALF和肺计数细胞。
    2. 在4℃下以400×g将肺细胞旋转5分钟,并将细胞以每50μl100万个细胞重悬于FACS缓冲液中。
    3. 在4℃下以400×g将BALF中的细胞分解5分钟。由于从气道中回收的细胞少得多,根据需要每50μlFACS缓冲液重新悬浮1-5×10 5个细胞。
    4. 在50μlFACS缓冲液中稀释0.25μgCD4-FITC和0.1μgCD16/32抗体(见附注4)。 
    5. 在FACS管中轻轻混合50μl细胞和50μl抗体。
    6. 在黑暗中孵育细胞15分钟,4℃
    7. 在4℃下,用2ml的FACS缓冲液,400℃洗涤细胞一次5分钟。
    8. 取出上清液,将细胞悬浮于100μlFACS缓冲液中
    9. 使用移液管将细胞通过70μm的细胞过滤器转移到新的FACS管中。使用流式细胞仪获取FACS数据并使用Flowjo软件分析数据(图2)。 (见注7)
    10. 流式细胞仪门控策略


      图2.区分气道,薄壁组织和肺血管中的CD4 + T细胞的门控策略。将记录CD4 + T细胞呼吸道,0.25μg荧光染料缀合的CD45和0.5μg荧光染料缀合的CD90.2抗体注射入经SARS-CoV Nulceocapsid蛋白的小鼠中和i.v.路线。然后收集气道和肺中的细胞,如实验程序中所述进行染色和收集,并使用Flowjo软件进行分析。 A.气道中的CD4 + T细胞:CD4 + CD45 CD90.2 。 B.实质中的CD4 + T细胞:CD4 + T细胞CD45细胞。脉管系统中的CD4 + sup细胞:CD4 +/CD40 + CD90 +。数据代表10个独立实验。 (Zhao等人,2016)

笔记

  1. CD45和CD90由所有CD4 + T细胞表达。 CD45和CD90抗体均可用于染色肺和循环中的CD4 + T细胞。优选选择藻红蛋白(PE),APC,BV(或其衍生物) - 共轭抗体,因为这些分子具有大的分子量,其将在体内标记期间最小化气道/血管渗漏。
  2. 确保将胶带穿在塑料干燥器格栅的中心孔上,因为小鼠有时能够挤压穿过该孔并跳入底部,填充异氟烷的室。
  3. 在这里,我们使用已经用表达SARS-CoV核衣壳蛋白的构建体免疫的抗原经验的小鼠(Zhao等人,2016)。该方案可用于研究小鼠肺中的记忆和效应物以及幼稚的CD4 + T细胞。
  4. 细胞还可以用其他表型(表面)抗体染色,或如前所述(Fett等人,2014))培养细胞内细胞因子染色。
  5. 对于表型染色,细胞将重新悬浮于FACS缓冲液中。对于细胞内染色,细胞将在完整的RPMI 1640培养基中重新配制
  6. 良好的灌注后,肺的外表面会变白。通常,5ml PBS是足够的。
  7. 该步骤是必要的,以防止流式细胞仪中的凝块形成。其他流式细胞仪,如LSR,Fortessa ...也可用于获取细胞。

食谱

  1. Avertin(250毫升)
    5g 2,2,2-三溴乙醇
    5ml 2-甲基-2-丁醇
    225毫升热纳米纯水(95℃)
    在通风柜中搅拌2小时,并将体积调节至250 ml
    0.2μm过滤器,等分并储存于-20°C
  2. 完成RPMI 1640培养基(500 ml)
    417毫升RPMI 1640培养基 12.5 ml 1 M HEPES
    5 ml 100x L-谷氨酰胺
    50ml FBS
    5 ml 100x MEM非必需氨基酸
    5 ml 100x丙酮酸钠
    5 ml 100x青霉素/链霉素
    0.5ml 2-巯基乙醇(50mM,在纳米纯H 2 O 2中,0.2μm过滤)
    储存于4°C
  3. 消化缓冲液(100ml)
    100毫克胶原酶D
    10毫克DNase I
    2 ml FBS
    1 ml 100x谷氨酰胺
    2.5 ml HEPES(1 M)
    1 ml 100x青霉素/链霉素
    使用HBSS将体积调整至100 ml 等分并储存于-20°C
  4. FACS缓冲液(500 ml) 1.7ml叠氮化钠(由纳米纯H 2 O制备的30%原料)
    15 ml FBS
    483.3 ml DPBS
    储存于4°C
  5. ACK裂解缓冲液
    1克KHCO 3
    8.3g NH 4 Cl
    37.2mg EDTA-Na 2
    用纳米纯H 2 O调节体积至1000毫升,并通过0.2微米过滤器过滤 在室温下存放

致谢

这项工作得到了"中国2015年千人计划奖"(J.Z.),广州市卫生联合创新重大项目(201604020011)(J.Z.)和N.I.H.的支持。 (USA)(PO1 AI060699,S.P.)。本文所述的方案基于以下手稿:Zhao等人。免疫。 2016年6月21日44(6):1379-91。

参考文献

  1. Anderson,KG,Sung,H.,Skon,CN,Lefrancois,L.,Deisinger,A.,Vezys,V.and Masopust,D。(2012)。切割边缘:血管内染色重新定义肺CD8 T细胞反应。 J Immunol 189 (6):2702-2706。
  2. Fett,C.,Zhao,J.and Perlman,S。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27390762"目标="_ blank">小鼠肺中CD8和CD4T细胞反应的测量。 4(6)。
  3. Zhao,J.,Zhao,J.,Mangalam,AK,Channappanavar,R.,Fett,C.,Meyerholz,DK,Agnihothram,S.,Baric,RS,David,CS和Perlman,S。(2016) ; 气道记忆CD4 + T细胞调解对新出现的呼吸道冠状病毒的保护性免疫。免疫性 44(6):1379-1391。
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引用:Wang, Y., Sun, J., Channappanavar, R., Zhao, J., Perlman, S. and Zhao, J. (2017). Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs. Bio-protocol 7(1): e2099. DOI: 10.21769/BioProtoc.2099.
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