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Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs
小鼠肺部的CD8和CD4 T淋巴细胞应激测定   

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Abstract

Study of the adaptive immune response to a viral challenge in an animal model often includes analysis of the T cell response. Here we discuss in detail the methods that are used to characterize the CD8 and CD4 T cell response following viral challenge in the lung.

Keywords: CD4 T cells(CD4 T细胞), CD8 T cells(CD8 T细胞), Coronavirus(冠状病毒), Lung infection(肺部感染), Lung preparation(肺的制备)

Materials and Reagents

  1. Mice (NCI) (BALB/c or C57BL/6, 4 weeks to 18+ months)
  2. Isofluorane (USP inhalation vapour, liquid) ( NDC: 57319-559-06 )
  3. Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, Gibco®, catalog number: 11965092 )
  4. Ketaset III ketamine HCl injection (USP 100 mg/ml) (DEA Schedule II Drug) (NDC: 0856-2013-01 )
  5. 100 mg/ml AnaSed injection xylazine (Lloyd Laboratories, NADA number: 139-236 )
  6. 0.9% sodium chloride irrigation (USP) (Baxter, catalog number: 2F7124 )
  7. 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Gibco®, catalog number: 14190-144 )
  8. Collagenase D (Roche Diagnostics, catalog number: 11088882001 )
  9. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  10. Hank’s balanced salt solution (HBSS) (Life Technologies, Gibco®, catalog number: 14025 )
  11. L-Glutamine (200 mM) (Life Technologies, Gibco®, catalog number: 25030-081 )
  12. Hepes (1 M) (Life Technologies, Gibco®, catalog number: 15630-080 )
  13. Penicillin streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  14. RPMI medium 1640 (Life Technologies, Gibco®, catalog number: 11875-093 )
  15. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250-100ML )
  16. BD Golgi PlugTM (BD, catalog number: 554722 )
  17. Normal rat serum (Life Technologies, InvitrogenTM, catalog number: 10710C )
  18. FITC Anti-mouse CD8a clone 53-6.7 (0.5 mg/ml) (BD, catalog number: 553031 )
  19. PerCP-cyanine5.5 Anti-mouse CD4 clone RM4-5 (eBioscience, catalog number: 45-0042-82 )
  20. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  21. Sodium azide (AMRESCO, catalog number: 0639 )
  22. BD Cytofix/CytopermTM fixation and permeabilization solution (BD, catalog number: 554722)
  23. BD Perm/WashTM buffer (BD, catalog number: 554722)
  24. APC Anti-mouse IFNγ clone XMG1.2 (eBioscience, catalog number: 17-7311-82 )
  25. Ketamine solution (see Recipes)
  26. Digestion buffer (see Recipes)
  27. RP10 (see Recipes)
  28. FACS buffer (see Recipes)
  29. Cell surface staining mixture (see Recipes)
  30. Intracellular staining mixture (see Recipes)

Equipment

  1. Dessicator (Narang Medical, catalog number: P37.1517P )
  2. Precision glide needle (25 G x 5/8) (BD, catalog number: 305122 )
  3. Gauze sponges (4 x 4 inch) (Pro Advantage® by NDC, catalog number: P157118 )
  4. 1 ml syringe (BD, catalog number: 309659 )
  5. 3 ml syringe (BD, catalog number: 309657 )
  6. Cell strainer (70 µm nylon) (BD, catalog number: 352350 )
  7. Tissue culture dishes (60 x 15 mm) (BD Biosciences, Falcon®, catalog number: 353002 )
  8. 96 well cell culture cluster (round bottom with Lid) (Corning, Costar®, catalog number: 3799 )
  9. 5 ml polystyrene round bottom tube (BD Biosciences, Falcon®, catalog number: 352054 )
  10. Absorbent pads (Covidien, catalog number: 949 )
  11. 10 ml syringe (BD, catalog number: 309604 )
  12. 12 well cell culture cluster (flat bottom with lid) (Corning, Costar®, catalog number: 3513 )
  13. 1 ml graduate transfer pipette (Research Products International, catalog number: 147501-1S )
  14. 15 ml screw cap tube conical (Sarstedt AG, catalog number: 62.554.002 )
  15. 50 ml screw cap tube conical (Sarstedt AG, catalog number: 62.547.004 )
  16. Biosafety hood
  17. Spray bottle with 70% ethanol
  18. Surgical scissors
  19. Polystyrene foam
  20. Pipetman p10
  21. Pipetman p200
  22. Pipetman p1000
  23. CO2 incubator
  24. Pipet aid
  25. Small metal weighing spatula
  26. Tweezers
  27. Vortexer
  28. Shaker rotisserie
  29. Refrigerated tabletop centrifuge
  30. Hemocytometer
  31. Flow cytometer

Procedure

  1. Infection
    1. Infect mice as described in Fett et al. (2014).

  2. Perfusion and removal of lungs
    1. Euthanize mice by intraperitoneal injection of 300 µl of ketamine/xylazine 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 ketamine/xylazine into the peritoneal cavity.
    2. When the mouse is fully anesthetized, immobilize it on a piece of polystyrene foam 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 abdominal and thoracic cavities with scissors by first making an incision from the lower abdominal to the throat of the animal. Opening the abdominal cavity exposes the underside of the diaphragm. Cut through the diaphragm with scissors and then remove the ribcage, fully exposing the heart and lungs.
    5. Fill a 10 ml syringe with sterile PBS and attach a 25 G x 5/8 needle. Insert the needle into the left ventricle of the heart and smoothly dispense PBS into the heart. While slowly dispensing PBS into the heart, use a tweezers to dissect the right atria away from the heart allowing blood to drain from circulation. Perfuse the general circulation with 5-7 ml of PBS. Remove the needle from the left ventricle and insert it into the right ventricle to more directly perfuse the lungs with the remaining 3-5 ml of PBS. Lungs without significant disease should perfuse easily.
    6. Cut the heart away from the lungs and then remove the lungs from the thoracic cavity after cutting the trachea and any remaining connective tissue.
    7. Place the lungs into the well of a 12 well tissue culture plate. If multiple lungs are to be harvested place lungs in PBS, store plate on ice and then transfer to a clean dry well immediately before processing.
    8. Mince the lungs into very fine pieces using a scissors.
    9. Transfer minced lung with a 1 ml transfer pipette to 5 ml of digestion buffer in a 15 ml conical tube.
    10. Place tubes on a shaker rotisserie and gently rotate at room temperature for 30 min.
    11. Place a 70 µm cell strainer into a 60 x 15 mm tissue culture dish.
    12. Transfer digestion buffer and lung tissue to the cell strainer using a 1 ml transfer pipette. Transfer some of the strained buffer into a 50 ml conical tube leaving enough liquid in the tissue culture dish so that the lung tissue on the screen of the cell strainer stays wet. With the flat end of a plunger from a 3 ml syringe gently press and dissociate tissue through strainer using small light movements. Intermittently flush tissue with PBS to move cells through the strainer. Process tissue until there is only connective tissue remaining on the strainer and transfer the remaining digestion buffer to the 50 ml conical tube.
    13. Spin down lung cells in 50 ml conical tubes for 10 min at 1,300 rpm at room temperature in a bucket tabletop centrifuge.
    14. Pour off supernatant and resuspend cells in 5 ml of RP10.

  3. Peptide stimulation and cell staining
    1. Count cells using a hemocytometer. Normal lung cell counts can vary from 3 million to 30 million or more depending on the stage and severity of the infection.
    2. Spin down cells for 5 min at 1,300 rpm.
    3. Resuspend cells in RP10 at 1 million cells per 100 µl.
    4. In a 96 well round bottom plate, add 100 µl of cells to 100 µl of RP10 containing Golgi Plug at a concentration of 2 µg/ml for a final concentration of 1 µg/ml. Wells should contain designated amount of peptide. Make sure to include wells for a no peptide control. In our SARS-CoV-infected samples, we routinely stimulate cells with peptide at a concentration of 1 µM. The SARS-CoV-specific peptides S366 (CD8; HNYKYRYL) and N353 (CD4; VNFNFNGL) are used to identify virus-specific CD8 and CD4 T cells by intracellular cytokine staining (ICS) for interferon gamma (IFN-γ). These two epitopes are H-2d-resticted and therefore recognized in BALB/c mice.
    5. Incubate cells at 37 °C, 5% CO2 for 5 h. Cells can then be stored overnight at 4 °C.

  4. Cell surface staining
    1. Spin down cells at 1,000 rpm for 5 min at 4 °C and then remove the supernatant by dumping the plate into a waste bin containing 10% bleach (when working with SARS-CoV) or rapidly flipping the plate onto paper towels.
    2. Add 100 µl of cell surface staining mixture per well. Mix thoroughly by pipeting each well up and down several times.
    3. Incubate in the dark for 15 min at 4 °C.
    4. Add 100 µl of FACS buffer and spin down cells at 1,300 rpm for 5 min at 4 °C. Remove supernatant. Add 100 µl of fixation and permeabilization solution; mix thoroughly by pipeting up and down several times.
    5. Incubate in the dark for 30 min at 4 °C.
    6. If working with SARS-CoV, cells can be transferred to a new 96 well round bottom plate and then removed from the BSL3.
    7. Using a pipetman, pass cells in fixation and permeablilization solution through a 70 µm cell strainer into FACS tubes.
    8. Resuspend cells in 2 ml of BD Perm/WashTM buffer, then spin down cells at 1,300 rpm for 5 min at 4 °C. Remove supernatant.

  5. Intracellular staining
    1. Resuspend cells in 100 µl of intracellular staining mixture per sample. Mix thoroughly by lightly vortexing tubes.
    2. Incubate in the dark for 30 min.
    3. Resuspend cells in at least 1 ml of FACS buffer then spin down cells at 1,300 rpm for 5 min at 4 °C. Remove supernatant.
    4. Resuspend cells in 100 µl FACS buffer.
    5. Acquire FACS data using a flow cytometer.


      Figure 1. Strategy for identifying SARS-CoV specific T cell responses. Mice were infected with mouse-adapted SARS-CoV. After 6-8 days, mice were euthanized, lungs removed and single cell suspensions prepared for flow cytometry. Left hand panel shows mononuclear cell gate. CD4 and CD8 T cells were identified and assessed for IFN-γ expression after stimulation with peptides.

Recipes

  1. Ketamine solution (10 ml)
    1.75 ml ketamine
    0.25 ml xylazine
    8 ml 0.9% sodium chloride irrigation
    Stored at room temperature
  2. Digestion buffer (100 ml)
    100 mg collagenase D
    10 mg DNase I
    2 ml FBS
    1 ml glutamine
    2.5 ml Hepes
    5 ml penicillin streptomycin
    89.5 ml HBSS
    Stored at -20 °C
  3. RP10 (500 ml)
    50 ml FBS
    500 µl 2-Mercaptoethanol (from 50 mM stock made in H2O)
    450 ml RPMI medium 1640
    Stored at 4 °C
  4. FACS buffer (500 ml)
    1.7 ml sodium azide (from 30% stock made in H2O)
    20 ml FBS
    480 ml PBS
    Stored at 4 °C
  5. Cell surface staining mixture (100 µl = 1 sample)
    1 µl rat serum
    0.25 µl CD4 PerCP-Cyanine5.5
    0.5 µl CD8a FITC
    100 µl FACS buffer
  6. Intracellular staining mixture (100 µl = 1 sample)
    0.3 µl IFNγ APC
    100 µl BD Perm/WashTM buffer

Acknowledgments

This work was supported by grants from the National Institutes of Health (PO1AI060699, RO1AI091322). The protocol described herein was based on the following manuscript: Zhao et al. (2010).

References

  1. Fett, C., DeDiego, M. L., Regla-Nava, J. A., Enjuanes, L. and Perlman, S. (2013). Complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking E protein. J Virol 87(12): 6551-6559.
  2. Fett, C., Zhao, J. C. and Perlman, S. (2014). Virus infection and titration of SARS-CoV in mouse lung. Bio-protocol 4(6): e1084.
  3. Netland, J., DeDiego, M. L., Zhao, J., Fett, C., Alvarez, E., Nieto-Torres, J. L., Enjuanes, L. and Perlman, S. (2010). Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease. Virology 399(1): 120-128.
  4. Zhao, J., Zhao, J. and Perlman, S. (2010). T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice. J Virol 84(18): 9318-9325.

简介

在动物模型中对病毒攻击的适应性免疫应答的研究通常包括T细胞应答的分析。 在这里我们详细讨论用于表征在肺中病毒攻击后的CD8和CD4T细胞应答的方法。

关键字:CD4 T细胞, CD8 T细胞, 冠状病毒, 肺部感染, 肺的制备

材料和试剂

  1. 小鼠(NCI)(BALB/c或C57BL/6,4周至18+月)
  2. 异氟烷(USP吸入蒸气,液体)(NDC:57319-559-06)
  3. Dulbecco's改良的Eagle培养基高葡萄糖(DMEM)(Life Technologies,Gibco ,目录号:11965092)
  4. Ketaset III氯胺酮HCl注射液(USP100mg/ml)(DEA Schedule II药物)(NDC:0856-2013-01)
  5. 100mg/ml注射甲苯噻嗪(Lloyd Laboratories,NADA号:139-236)
  6. 0.9%氯化钠灌注(USP)(Baxter,目录号:2F7124)
  7. 1×Dulbecco磷酸盐缓冲盐水(DPBS)(Life Technologies,Gibco ,目录号:14190-144)
  8. 胶原酶D(Roche Diagnostics,目录号:11088882001)
  9. DNase I(Roche Diagnostics,目录号:10104159001)
  10. Hank's平衡盐溶液(HBSS)(Life Technologies,Gibco ,目录号:14025)
  11. L-谷氨酰胺(200mM)(Life Technologies,Gibco ,目录号:25030-081)
  12. Hepes(1M)(Life Technologies,Gibco ,目录号:15630-080)
  13. 青霉素链霉素(Life Technologies,Gibco ,目录号:15140-122)
  14. RPMI培养基1640(Life Technologies,Gibco ,目录号:11875-093)
  15. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250-100ML)
  16. BD Golgi Plug TM (BD,目录号:554722)
  17. 正常大鼠血清(Life Technologies,Invitrogen TM,目录号:10710C)
  18. FITC抗小鼠CD8a克隆53-6.7(0.5mg/ml)(BD,目录号:553031)
  19. PerCP-花青5.5抗小鼠CD4克隆RM4-5(eBioscience,目录号:45-0042-82)
  20. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150)
  21. 叠氮化钠(AMRESCO,目录号:0639)
  22. BD Cytofix/Cytoperm TM 固定和透化溶液(BD,目录号:554722)
  23. BD Perm/Wash TM 缓冲液(BD,目录号:554722)
  24. APC抗小鼠IFNγ克隆XMG1.2(eBioscience,目录号:17-7311-82)
  25. 氯胺酮溶液(见配方)
  26. 消化缓冲液(参见配方)
  27. RP10(参见配方)
  28. FACS缓冲区(参见配方)
  29. 细胞表面染色混合物(见配方)
  30. 细胞内染色混合物(见配方)

设备

  1. Dessicator(Narang Medical,目录号:P37.1517P)
  2. 精密滑动针(25G×5/8)(BD,目录号:305122)
  3. 纱布海绵(4×4英寸)(由NDC的Pro Advantage ,目录号:P157118)
  4. 1ml注射器(BD,目录号:309659)
  5. 3ml注射器(BD,目录号:309657)
  6. 细胞过滤器(70μm尼龙)(BD,目录号:352350)
  7. 组织培养皿(60×15mm)(BD Biosciences,Falcon ,目录号:353002)
  8. 96孔细胞培养物簇(带有盖的圆底)(Corning,Costar ,目录号:3799)
  9. 5ml聚苯乙烯圆底管(BD Biosciences,Falcon ,目录号:352054)
  10. 吸收垫(Covidien,目录号:949)
  11. 10ml注射器(BD,目录号:309604)
  12. 12孔细胞培养物簇(带盖的平底)(Corning,Costar ,目录号:3513)
  13. 1ml研磨转移移液管(Research Products International,目录号:147501-1S)
  14. 15ml螺旋盖管锥形(Sarstedt AG,目录号:62.554.002)
  15. 50ml锥形螺旋盖管(Sarstedt AG,目录号:62.547.004)
  16. 生物安全罩
  17. 喷雾瓶用70%乙醇
  18. 外科剪刀
  19. 聚苯乙烯泡沫
  20. Pipetman p10
  21. Pipetman p200
  22. Pipetman p1000
  23. CO <2>孵化器
  24. 移液助剂
  25. 小金属称重刮刀
  26. 镊子
  27. Vortexer
  28. 振动器旋床
  29. 冷冻式台式离心机
  30. 血细胞计数器
  31. 流式细胞仪

程序

  1. 感染
    1. 感染小鼠,如Fett等人 (2014)。

  2. 灌注和去除肺
    1. 安乐死小鼠通过腹膜内注射300微升氯胺酮/赛拉嗪溶液。将鼠标固定在手掌中,水平握住它,然后以与小鼠相切的浅角插入针头,慢慢地将氯胺酮/赛拉嗪分配到腹膜腔中。
    2. 当鼠标完全麻醉时,通过用25 G×5/8针固定每个肢体将其固定在一块聚苯乙烯泡沫上。
    3. 使用装满70%乙醇的喷雾瓶润湿小鼠的毛皮
    4. 用剪刀切开腹腔和胸腔,首先从下腹部到动物的喉咙切开。打开腹腔暴露隔膜的下侧。用剪刀切开隔膜,然后取下胸腔,完全暴露心脏和肺
    5. 用无菌PBS填充10ml注射器,并附上25G×5/8针。将针插入心脏的左心室,并顺利地将PBS分配到心脏。在慢慢地将PBS分配到心脏中的同时,使用镊子将右心房从心脏分离,从而允许血液从循环中排出。用5-7ml PBS灌注一般循环。从左心室取出针头,将其插入右心室,更直接灌注肺与剩余的3-5毫升PBS。没有显着疾病的肺应该容易灌注。
    6. 切开心脏远离肺部,然后在切除气管和任何残留的结缔组织后从胸腔中取出肺。
    7. 将肺放入12孔组织培养板的孔中。 如果要收获多个肺,在PBS中放置肺,将板在冰上存储,然后在处理之前立即转移到干净的干井。
    8. 用剪刀将肺切成很细的碎片。
    9. 在15ml锥形管中用1ml转移移液管将切碎的肺转移至5ml消化缓冲液中。
    10. 将管放置在振荡器旋转式烤架上,并在室温下轻轻旋转30分钟。
    11. 将70微米的细胞过滤器放入60×15毫米组织培养皿。
    12. 转移消化缓冲液和肺组织到细胞过滤器使用1毫升移液管。将一些应变缓冲液转移到50ml锥形管中,在组织培养皿中留下足够的液体,使得细胞滤网的筛网上的肺组织保持湿润。使用来自3ml注射器的柱塞的平端,使用小的光运动轻轻地按压并解离组织通过过滤器。用PBS间歇冲洗组织,以移动细胞通过过滤器。处理组织,直到只有结缔组织保留在过滤器上,并将剩余的消化缓冲液转移到50ml锥形管。
    13. 在桶式台式离心机中在室温下以1,300rpm将50ml锥形管中的肺细胞旋转10分钟。
    14. 倒出上清液并将细胞重悬于5ml RP10中
  3. 肽刺激和细胞染色
    1. 使用血细胞计数器计数细胞。正常肺细胞计数可以从3百万到3千万或更多,这取决于感染的阶段和严重程度。
    2. 以1,300rpm速度旋转细胞5分钟。
    3. 在RP10中重悬细胞,每100μl100万个细胞
    4. 在96孔圆底板中,将100μl细胞加入到含有高尔基塞的RP10的100μl中,浓度为2μg/ml,终浓度为1μg/ml。孔应含有指定量的肽。确保包括无肽对照的孔。在我们的SARS-CoV感染的样品中,我们常规地用浓度为1μM的肽刺激细胞。 SARS-CoV特异性肽S366(CD8; HNYKYRYL)和N353(CD4; VNFNFNGL)用于通过干扰素γ(IFN-γ)的细胞内细胞因子染色(ICS)鉴定病毒特异性CD8和CD4T细胞。这两个表位是H-2抗原决定簇,因此在BALB/c小鼠中被识别。
    5. 在37℃,5%CO 2孵育细胞5小时。然后将细胞在4℃下保存过夜。

  4. 细胞表面染色
    1. 在4℃下以1,000rpm旋转细胞5分钟,然后通过将平板倾倒入含有10%漂白剂(当使用SARS-CoV)或将片快速翻转到纸巾上的废物箱中来除去上清液。
    2. 每孔加入100微升细胞表面染色混合物。 通过将每个孔向上和向下移动几次来彻底混合
    3. 在4℃下避光孵育15分钟。
    4. 加入100μlFACS缓冲液,并在4℃下以1,300rpm离心细胞5分钟。 除去上清液。 加入100μl固定和透化溶液; 通过上下吹打数次彻底混合
    5. 在4℃下避光孵育30分钟。
    6. 如果使用SARS-CoV,细胞可以转移到新的96孔圆底板,然后从BSL3中取出。
    7. 使用移液管,将固定和渗透溶液中的细胞通过70微米的细胞过滤器进入FACS管。
    8. 重悬细胞在2ml的BD Perm/Wash TM缓冲液中,然后在4℃下以1,300rpm离心细胞5分钟。 除去上清液。

  5. 细胞内染色
    1. 重悬细胞在100微升细胞内染色混合物每个样品。 轻轻涡旋试管充分混合
    2. 在黑暗中孵育30分钟。
    3. 重悬细胞在至少1毫升FACS缓冲液然后旋转细胞在1,300 rpm 5分钟在4℃。除去上清液。
    4. 重悬细胞在100微升FACS缓冲液
    5. 使用流式细胞仪获取FACS数据

      图1.用于鉴定SARS-CoV特异性T细胞应答的策略。用小鼠适应的SARS-CoV感染小鼠。 6-8天后,将小鼠安乐死,取出肺并制备用于流式细胞术的单细胞悬浮液。左侧面板显示单核细胞门。 CD4和CD8 T细胞被鉴定并评估IFN- size:12.0pt; font-family:Symbol;"> 食谱

      1. 氯胺酮溶液(10ml) 1.75 ml氯胺酮 0.25ml甲苯胺
        8毫升0.9%氯化钠灌注
        在室温下贮存
      2. 消化缓冲液(100ml)
        100mg胶原酶D
        10 mg DNase I
        2 ml FBS
        1 ml谷氨酰胺
        2.5 ml Hepes
        5ml青霉素链霉素 89.5ml HBSS
        储存于-20°C
      3. RP10(500ml) 50ml FBS
        500μl2-巯基乙醇(从50mM在H 2 O中制备的储备液) 450ml RPMI培养基1640
        储存在4°C
      4. FACS缓冲液(500ml) 1.7ml叠氮化钠(从H 2 O中制备的30%原液) 20ml FBS
        480 ml PBS
        储存在4°C
      5. 细胞表面染色混合物(100μl= 1个样品)
        1μl大鼠血清
        0.25μlCD4 PerCP-Cyanine5.5
        0.5μlCD8a FITC
        100μlFACS缓冲液
      6. 细胞内染色混合物(100μl= 1个样品)
        0.3μlIFNγAPC
        100μlBD Perm/Wash TM 缓冲液

      致谢

      这项工作是由国家卫生研究院的拨款支持(PO1AI060699,RO1AI091322)。 本文所述的方案基于以下手稿:Zhao等人(2010)。

      参考文献

      1. Fett,C.,DeDiego,M.L.,Regla-Nava,J.A.,Enjuanes,L.and Perlman,S。(2013)。 通过用小鼠免疫在老年小鼠中完全保护免受严重急性呼吸综合征冠状病毒介导的致死性呼吸道疾病 - 适应缺乏E蛋白的病毒。 Virol 87(12):6551-6559。
      2. Fett,C.,Zhao,J.C。和Perlman,S。(2014)。 小鼠肺中的SARS-CoV的病毒感染和滴定。生物协议 4(6):e1084。
      3. Netland,J.,DeDiego,M.L.,Zhao,J.,Fett,C.,Alvarez,E.,Nieto-Torres,J.L.,Enjuanes,L.and Perlman, 在E蛋白中缺失的减毒性严重急性呼吸综合征冠状病毒的免疫保护免于致命的呼吸道疾病。 a> Virology 399(1):120-128。
      4. Zhao,J.,Zhao,J.and Perlman,S。(2010)。 T细胞应答对于严重急性呼吸综合征冠状病毒感染的小鼠中的临床疾病和病毒清除是必需的。 84(18):9318-9325。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fett, C., Zhao, J. and Perlman, S. (2014). Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs. Bio-protocol 4(6): e1083. DOI: 10.21769/BioProtoc.1083.
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