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Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia
采用流式细胞仪检测被带有荧光报告基因的曲霉(FLARE)分生孢子感染过的小鼠肺部和支气管肺泡灌洗液细胞

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Abstract

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.

Keywords: Fungus(真菌), Microscopy(显微镜), Immunity(免疫), Lung(肺), Reporter(记者)

Materials and Reagents

  1. Cell strainers - 40 μm and 100 μm
  2. Syringes
  3. Injector (Modified precision dispensing tips) (Nordson, catalog number: 7018166 )
  4. 15 ml tubes
  5. 96-well plate , U-bottom
  6. Mice: C57BL/6 (Jackson Laboratories, catalog number: 000664 )
  7. Fungal strains (Af293 and Af293-DsRed) (Jhingran et al., 2012)
  8. FATAL-plus solution (Vortech Pharmaceuticals Ltd, National Drug Code: 0298-9373-68 )
  9. BD BBLTM Sabouraud dextrose agar (Emmons), slants (pH 6.9) (100/sp) (BD, catalog number: 221827 )
  10. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  11. Tween 20
  12. Tris base
  13. NaHCO3
  14. Na2CO3
  15. Biotin-XX, SSE (6-((6-((biotinoyl)amino)hexanoyl)amino) hexanoic acid, sulfosuccinimidyl ester, sodium salt) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: B6352 )
  16. DMSO
  17. Streptavidin, Alexa Fluor® 633 conjugate (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S-21375 )
  18. IsothesiaTM, Isoflurane (Henry Schein Animal Health, SKU number: 0 29405 )
  19. RBC lysis buffer (Biolegend, catalog number: 420301 )
  20. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  21. Collagenase type IV (Worthington Biochemical, catalog number: LS004189 )
  22. EDTA
  23. Mouse BD Fc BlockTM (BD, BD PharmingenTM, catalog number: 553142 )
  24. Antibodies (listed in Procedure section)
  25. PBS Tween (0.025%) (see Recipes)
  26. Tris-chloride (1 M, pH 8) (see Recipes)
  27. Sodium bicarbonate buffer (0.1 M, pH 9.5) (see Recipes)
  28. Lung digestion buffer (for each lung) (see Recipes)
  29. FACS buffer (see Recipes)

Equipment

  1. Hemocytometer
  2. Microscope
  3. Centrifuge
  4. Plexiglass stand
  5. Dissection scissors and forceps
  6. Bronchoalveolar lavage fluid (BALF) catheters (Exel International, catalog number: 26738 )
  7. 4-way large bore (lipid resistant) stopcock with rotating male luer lock adapter (Baxter, catalog number: 2C6204 )
  8. gentleMACS tissue dissociator, MACSMix tube rotator and C tubes (Miltenyi Biotech)
  9. Automated cell counter or hemocytometer
  10. Flow cytometer capable of analyzing at least 7 fluorescent parameters (such as BD LSRII). The 532 or 561 nm (but not 488 nm) laser excites DsRed fluorescence and the 633 nm laser excites Alexa Fluor 633 fluorescence.

Procedure

  1. Preparation of FLARE conidia
    1. Harvest conidia from 7-10 days old slants (Sabouraud dextrose agar slants) grown at 37 °C by adding 10 ml ice-cold PBST (0.025% Tween-20 in PBS) and pipetting multiple times with gentle scrapping.
      Note: Maintain conidia at 4 °C or on ice for all subsequent steps unless otherwise stated. All infection and animal studies need to be performed following biosafety level 2 (BSL2).
    2. Filter the conidial suspension sequentially through a 100 μm and a 40 μm cell strainer to eliminate hyphal fragments. Centrifuge the filtered suspension at 400 x g for 5 min. Discard the supernatant and resuspend the conidial pellet in 10 ml PBST.
    3. Count the conidia using a hemocytometer and adjust the final concentration to 5-6 x 108 conidia per ml.
    4. Centrifuge desired amount of conidial suspension at 400 x g for 5 min, discard supernatant and resuspend the pellet in 50 mM NaHCO3 (pH 9.5) maintaining the final concentration as 5 x 108 conidia per ml.
    5. Add 0.5 mg/ml Biotin-XX, SSE (6-((6-((biotinoyl)amino)hexanoyl)-amino) hexanoic acid), sulfosuccinimidyl ester, sodium salt (stock 50 mg/ml in DMSO) to the conidial suspension and incubate with rotation in the dark.
      Note: A minimum of 2 h incubation is recommended for efficient biotinylation. This step can be extended for up to 12 h (O/N incubation). The tubes containing biotinylated conidia should be covered with aluminum foil at all times.
    6. Centrifuge and decant the supernatant. Neutralize unbound biotin by resuspending the pellets and incubating conidia in 0.1 M Tris-HCl (pH 8.0) for 30 min with constant rotation.
    7. Repeat step A6 and resuspend the biotinylated conidia in (indicate volume) PBS containing 0.02 mg/ml AF633-streptavidin (stock 2 mg/ml in PBS). Incubate for 30 min at RT with constant rotation.
    8. Wash once with PBST and adjust the concentration to 6 x 107 per ml for intratracheal challenge.
      Note: Check the labeling efficiency by flow cytometry before using the FLARE conidia for experiments (Figure 1).


      Figure 1. Labeling efficiency of FLARE conidia. Flow plots depict fluorescence intensity of (A) unlabeled, (B) AF633-labeled, (C) DsRed-labeled, and (D) FLARE conidia in AF633 (X-axis) and DsRed (Y-axis) channels using a BD LSR II flow cytometer equipped with 532 nm and 633 nm lasers.

  2. Murine intratracheal challenge with FLARE conidia
    1. Adjust conidial suspension at 6 x 108/ml. Load a 1 ml syringe fitted with a curved blunt-ended, 20 gauge precision tip (Figure 2; lower syringe) with 50 μl conidial suspension (typical inoculum: 3 x 107 conidia) and expel any air bubbles. The inoculum volume should not exceed 75 μl.


      Figure 2. Curved blunt-ended 20 G tip used to administer conidia intratracheally. The injection syringe (lower syringe) was fabricated from a straight 20 G syringe (upper syringe) by applying heat via a Bunsen burner.

    2. Anaesthetize mice using an isoflurane unit that pumps a mixture of isoflurane (3.5%, vol/vol) and oxygen in an anesthesia chamber.
    3. Confirm anesthesia by monitoring breathing rate and by toe pinch. Once anesthetized, immobilize the mouse in an upright position on an angled plexiglass stand by hooking its incisors onto a rubber band. Secure the mouse torso with a second rubber band (Figure 3).


      Figure 3. Anaesthetized mouse that is immobilized in preparation for intratracheal administration of conidia

    4. Gently grasp the tongue on one side (typically right hand) using forceps and then grasp tongue with gloved left thumb and forefinger, opening the jaw and flexing the jaw forward. With the right hand guide the syringe containing inoculum over the tongue towards its trachea until a hub of the catheter has been inserted into oral cavity (Figure 4). Push the plunger to deliver inoculum and withdraw the syringe quickly to avoid suffocation. There should be no resistance to delivering the inoculum.
       Note: A video demonstration of murine intratracheal administration can be seen at Hasenberg et al. (2011).


      Figure 4. Intratracheal administration of conidia

    5. Remove the mouse from the plexiglass stand and place it back into its original cage. Monitor the mouse until it starts moving and then proceed to the next mouse. Once all mice are inoculated and regained conscious, return the cage to the rack.

  3. Tissue harvest
    1. Euthanize mice by injecting sodium pentobarbital solution, 300 mg/kg body weight via the intraperitoneal route. When the mouse is euthanized (4-6 min), exsanguinate the animal by making an incision in abdominal aorta to minimize blood contamination during BAL procedures.
    2. Expose trachea by dissecting out tissues from neck and make a minute incision into the trachea to allow the passage of 18 G lavage catheter through it (Figure 5).
    3. Insert a BALF catheter into the mouse trachea and repeatedly inject 0.5 ml of ice cold PBS/5% FBS (from the “input” syringe filled with 3 ml of PBS/5% FBS) and then aspirate BALF from the inflated lungs into the initially unfilled “output” syringe via a 3-way stop cock. Typically, we inject a total of 3 ml and recover 2.5 ml of BALF in the output syringe. The 3-way stopcock is adjusted after each injection and aspiration step to ensure that BALF is captured in the output syringe.
    4. Transfer BALF into empty tubes and store on ice.
    5. Collect lungs in tubes containing 2 ml ice cold PBS and store on ice.


      Figure 5. BALF harvest using a 3-way stopcock

  4. Lung processing
    1. Transfer lungs into gentleMACS C tubes each containing 5 ml of freshly prepared lung digestion buffer.
    2. Attach the tubes onto the gentleMACS dissociator and run program “m_lung_01” to dissociate lung samples. Incubate the tubes for 45 min at 37 °C on the MACS mix tube rotator to allow digestion.
    3. Bring the tubes back to the gentleMACS dissociator and run program “m_lung_02, which completes preparation of single cell suspension of lung samples.
    4. Briefly spin tubes (300 x g for 1 min) to bring cells at the bottom and using a pipette, transfer the contents into fresh 15 ml tubes.
      Note: All subsequent steps are to be performed at 4 °C unless otherwise mentioned.
    5. Centrifuge the tubes at 300 x g for 5 min, discard supernatant and lyse RBCs by resuspending pellet in 1 ml of 1x RBC lysis buffer for 5 min at RT.
    6. Following RBC lysis, add 9 ml of ice cold 5% FBS/PBS to each tube and pass the entire 10 ml lung suspension through a 100 μm cell strainer into fresh 15 ml tubes.
    7. Centrifuge tubes, discard supernatant and resuspend cells in 2 ml ice cold 5% FBS/PBS.
    8. Count cells in an automated cell counter, transfer 200 μl cell suspension (approx. 2-5 million cells) in a U-bottom, 96-well plate and prepare for staining with fluorescent antibodies.

  5. BALF processing
    1. Spin tubes containing BALF at 300 x g for 5 min, discard supernatant and transfer the pellets in a U-bottom, 96-well plate for staining with fluorescent antibodies.

  6. Flow cytometry of lung and BALF cell suspension
    1. Resuspend the lung and BALF samples in 50 μl of FACS buffer containing Fc block in 1:100 dilution, incubate at 4 °C for 10 min.
    2. Wash the cells once in FACS buffer and add 50 μl of stain cocktail mix containing antibodies (purchased from ebiosciences/BD/AbD serotec) listed below:
      1. Ab staining cocktail for lung neutrophils and Ly6Chi monocytes (Figure 6)
        CD45.2 (clone 104) - PerCP-Cy5.5
        Ly6G (1A8) - FITC
        Ly6C (clone AL-21) - PE-Cy7
        Ly6B.2 (clone 7/4) - AF700
        CD11b (clone M1/70) - Pacific Blue
        Neutrophils: CD45.2+Ly6G+Ly6CloCD11b+Ly6B.2+
        Monocytes: CD45.2+Ly6G-Ly6ChiCD11b+Ly6B.2+


        Figure 6. Gating strategy to measure conidial uptake and killing by Ly6Chi monocytes and neutrophils in lung tissue. In the right column, the red gate consists of leukocytes with live conidia, the blue gate leukocytes with dead conidia, and the black gate bystander leukocytes that contain no conidia.

      2. Ab staining cocktail for lung macrophages and CD11b+ DCs (Figure 7)
        CD45.2 (clone 104) - PerCP-Cy5.5
        CD103 (2E7) - FITC
        CD11c (HL3) - PE-Cy7
        MHC Class II (M5/114.15.2) - AF700
        CD11b (clone M1/70) - Pacific Blue
        Macrophages: CD45.2+CD11c+MHCIIvar Autofluorescent cells
        CD11b+ DCs: CD45.2+CD11c+MHCIIvar CD103-CD11b+


        Figure 7. Gating strategy to measure conidial uptake and killing by CD11b+ DCs and macrophages in lung tissue. In the right column, the red gate consists of leukocytes with live conidia, the blue gate leukocytes with dead conidia, and the black gate bystander leukocytes that contain no conidia.

      3. Ab staining cocktail for BALF neutrophils and alveolar macrophages (Figure 8)
        CD45.2 (clone 104) - PerCP-Cy5.5
        Ly6G (1A8) - FITC
        CD11c (HL3) - PE-Cy7
        Ly6B.2 (clone 7/4) - AF700
        CD11b (clone M1/70) - Pacific Blue
        Neutrophils: CD45.2+Ly6G+CD11c-CD11b+Ly6B.2+
        Alveolar macrophages: CD45.2+Ly6G- CD11b-Ly6B.2-CD11c+
        Note: Leave the AF633 and DsRed channels open in all FLARE experiments. Leukocytes that are AF633+DsRed+ harbor live conidia, leukocytes that are AF633+DsRed- harbor dead conidia and leukocytes that are AF633-DsRed- represent bystander population (Jhingran et al., 2012).


        Figure 8. Gating strategy to measure conidial uptake and killing by BALF neutrophils and alveolar macrophages. In the right column, the red gate consists of leukocytes with live conidia, the blue gate leukocytes with dead conidia, and the black gate bystander leukocytes that contain no conidia.

    3. Incubate in dark at 4 °C for 20 min. Wash once, resuspend in FACS buffer and analyze with flowcytometry within 1 h. We do not recommend fixing samples as we observed DsRed fluorescent intensity was significantly lower if samples were fixed with 1% PFA and analyzed 24 h later.
      Recommended band-pass filter setting:
      DsRed (PE): 586/15
      PerCP-Cy5.5: 695/40
      FITC: 530/30
      PE-Cy7: 780/60
      AF700: 720/40
      Pacific Blue: 450/50
      AF633 (APC): 660/20

Recipes

  1. PBS Tween (0.025%)
    0.025% Tween 20 (v/v, 2.5 ml from 10% stock)
    1,000 ml PBS (pH 7.4)
    Filter sterilize and store at 4 °C
  2. Tris-chloride (1 M, pH 8)
    121.14 g Tris base
    Dissolve in 800 ml Milli Q water
    Adjust pH to 8.0 using 10 N HCl
    Adjust volume to 1,000 ml and store at 4 °C
  3. Sodium bicarbonate buffer (0.1 M, pH 9.5)
    8.40 g NaHCO3
    3.56 g Na2CO3
    Dissolve in 1,000 ml Milli Q water and store at 4 °C
  4. Lung digestion buffer (for each lung)
    5 ml 5% FBS/PBS (pH 7.4)
    2.2 mg/ml collagenase type IV
    100 μg/ml DNase I
  5. FACS buffer
    0.5% BSA (2.5 g)
    0.1 mM EDTA (100 μl from 0.5 M stock)
    500 ml 1x PBS (pH 7.4)
    Store at 4 °C

Acknowledgments

The studies were performed with support from the following funding agencies and grants: The Lucille Castori Center for Microbes, Inflammation, and Cancer (CMIC) postdoctoral fellowship to A.J., NIH grants R01 AI093808 and R21 AI105617 to T.M.H. T.M.H. is an Investigator in the Pathogenesis of Infectious Diseases supported by the Burroughs Wellcome Fund. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.

References

  1. Espinosa, V., Jhingran, A., Dutta, O., Kasahara, S., Donnelly, R., Du, P., Rosenfeld, J., Leiner, I., Chen, C. C., Ron, Y., Hohl, T. M. and Rivera, A. (2014). Inflammatory monocytes orchestrate innate antifungal immunity in the lung. PLoS Pathog 10(2): e1003940.
  2. Hasenberg, M., Kohler, A., Bonifatius, S., Jeron, A. and Gunzer, M. (2011). Direct observation of phagocytosis and NET-formation by neutrophils in infected lungs using 2-photon microscopy. J Vis Exp (52).
  3. Heung, L. J., Jhingran, A. and Hohl, T. M. (2015). Deploying FLAREs to visualize functional outcomes of host-pathogen encounters. PLoS Pathog 11(7): e1004912.
  4. Jhingran, A., Mar, K. B., Kumasaka, D. K., Knoblaugh, S. E., Ngo, L. Y., Segal, B. H., Iwakura, Y., Lowell, C. A., Hamerman, J. A., Lin, X. and Hohl, T. M. (2012). Tracing conidial fate and measuring host cell antifungal activity using a reporter of microbial viability in the lung. Cell Rep 2(6): 1762-1773.

简介

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.

关键字:真菌, 显微镜, 免疫, 肺, 记者

材料和试剂

  1. 细胞过滤器 - 40μm和100μm
  2. 注射器
  3. 注射器(改进的精密分配尖端)(Nordson,目录号:7018166)
  4. 15 ml管
  5. 96孔板,U底
  6. 小鼠:C57BL/6(Jackson Laboratories,目录号:000664)
  7. 真菌菌株(Af293和Af293-DsRed)(Jhingran等人,2012)
  8. FATAL-plus溶液(Vortech Pharmaceuticals Ltd,国家药品代码:0298-9373-68)
  9. BD BBL Sabouraud右旋糖琼脂(Emmons),斜面(pH 6.9)(100/sp)(BD,目录号:221827)
  10. PBS(Thermo Fisher Scientific,Gibco TM ,目录号:14190-144)
  11. 吐温20
  12. Tris碱
  13. NaHCO 3
  14. Na 2 3
  15. 生物素-XX,SSE(6 - ((6 - ((生物素酰基)氨基)己酰基)氨基)己酸,磺基琥珀酰亚胺酯,钠盐)(Thermo Fisher Scientific,Molecular Probes,目录号: B6352)
  16. DMSO
  17. 链霉亲和素,Alexa Fluor 633缀合物(Thermo Fisher Scientific,Molecular Probes TM,目录号:S-21375)
  18. Isothesia ,异氟烷(Henry Schein Animal Health,SKU编号:029405)
  19. RBC裂解缓冲液(Biolegend,目录号:420301)
  20. DNase I(Roche Diagnostics,目录号:10104159001)
  21. 胶原酶IV型(Worthington Biochemical,目录号:LS004189)
  22. EDTA
  23. 小鼠BD Fc Block TM (BD,BD Pharmingen TM ,目录号:553142)
  24. 抗体(在程序部分列出)
  25. PBS Tween(0.025%)(参见配方)
  26. Tris-chloride(1M,pH 8)(参见配方)
  27. 碳酸氢钠缓冲液(0.1M,pH 9.5)(见配方)
  28. 肺消化缓冲液(每只肺)(见配方)
  29. FACS缓冲液(见配方)

设备

  1. 血细胞计数器
  2. 显微镜
  3. 离心机
  4. 有机玻璃支架
  5. 解剖剪刀和镊子
  6. 支气管肺泡灌洗液(BALF)导管(Exel International,目录号:26738)
  7. 带旋转阳路厄锁紧适配器(Baxter,目录号:2C6204)的4通大口径(抗脂)旋塞阀
  8. 温和的MACS组织分离器,MACSMix管旋转器和C管(Miltenyi Biotech)
  9. 自动细胞计数器或血细胞计数器
  10. 能够分析至少7个荧光参数(例如BD LSRII)的流式细胞仪。 532或561nm(但不是488nm)激光激发DsRed荧光,633nm激光激发Alexa Fluor 633荧光。

程序

  1. FLARE分生孢子的制备
    1. 通过加入10ml冰冷的PBST(PBS中的0.025%Tween-20)并轻轻刮去多次,在37℃下生长7-10天龄的斜面(Sabouraud葡萄糖琼脂斜面)的分生孢子。
      注意:除非另有说明,否则所有后续步骤应在4°C或冰上保存分生孢子。所有感染和动物研究需要在生物安全2级(BSL2)后进行。
    2. 依次通过100μm和40μm细胞过滤器过滤分生孢子悬浮液以消除菌丝碎片。在400×g离心过滤的悬浮液5分钟。弃去上清液并将分生孢子沉淀重悬在10ml PBST中。
    3. 使用血细胞计数器计数分生孢子,并将最终浓度调整为每毫升5-6×10 8个分生孢子。
    4. 在400×g离心所需量的分生孢子悬浮液5分钟,弃去上清液并将沉淀物重悬在50mM NaHCO 3(pH 9.5)中,保持终浓度为5×10 5 分生孢子/ml。
    5. 加入0.5mg/ml生物素-XX,SSE(6 - ((6 - ((生物素酰基)氨基)己酰基) - 氨基)己酸),磺基琥珀酰亚胺酯钠盐(DMSO中50mg /并旋转孵育 黑暗。
      注意:建议至少进行2小时孵育以进行有效的生物素化。该步骤可以延长长达12小时(O/N孵育)。含有生物素化分生孢子的试管应始终用铝箔覆盖。
    6. 离心和倾析上清液。通过重悬沉淀物并在0.1M Tris-HCl(pH 8.0)中恒定旋转孵育分生孢子30分钟来中和未结合的生物素。
    7. 重复步骤A6,并将生物素化的分生孢子悬浮在含有0.02mg/ml AF633-链霉亲和素(PBS中的储备液2mg/ml)的(指示体积)PBS中。在室温下恒定旋转孵育30分钟。
    8. 用PBST洗涤一次,并调整浓度至6×10 7/ml,用于气管内攻击。
      注意:在使用FLARE分生孢子进行实验之前,通过流式细胞术检查标记效率(图1)。


      图1. FLARE分生孢子的标记效率。流动图描绘了(A)未标记的,(B)AF633-标记的,(C)DsRed-标记的和使用装备有532nm和633nm激光的BD LSR II流式细胞仪在AF633(X轴)和DsRed(Y轴)通道中的分生孢子。
  2. 鼠气管内挑战与FLARE分生孢子
    1. 调节6×10 8/ml/ml的分生孢子悬浮液。加载配有弯曲的钝端,20规格精密尖端(图2;下注射器)和50μl分生孢子悬浮液(典型接种:3×10 7分生孢子)的1ml注射器,并排出任何空气气泡。接种量不应超过75μl

      图2.用于气管内施用分生孢子的弯曲的钝端20G尖端。通过经由本生灯施加热量,从直的20G注射器(上注射器)制造注射器(下注射器)。

    2. 使用异氟烷单元麻醉小鼠,该麻醉单元在麻醉室中泵送异氟烷(3.5%,体积/体积)和氧气的混合物。
    3. 通过监测呼吸频率和脚趾确认麻醉。一旦麻醉,通过将其门牙钩在橡皮筋上,将小鼠以直立位置固定在倾斜的有机玻璃支架上。用第二个橡皮筋固定鼠标躯干(图3)。


      图3.固定化用于气管内给予分生孢子的麻醉小鼠

    4. 使用镊子轻轻抓住一侧(通常是右手)的舌头,然后用手套左拇指和食指抓住舌头,打开颌骨和向前颚屈曲。用右手将包含接种物的注射器在舌头上朝向其气管引导,直到导管的毂已经插入口腔(图4)。推动柱塞以递送接种物并迅速撤回注射器以避免窒息。应该没有阻碍递送接种物。
      注意:在Hasenberg等人的文章中可以看到鼠气管内给药的视频演示。 (2011)。


      图4.气管内给予分生孢子

    5. 从有机玻璃支架上取下鼠标,并将其放回原来的笼子里。监视鼠标,直到它开始移动,然后继续下一个鼠标。一旦所有小鼠接种和 恢复意识,将笼子放回架子
  3. 组织收获
    1. 通过腹膜内途径注射300mg/kg体重的戊巴比妥钠溶液安乐死小鼠。当小鼠安乐死(4-6分钟)时,通过在腹主动脉切开以在BAL程序期间使血液污染最小化来放弃动物。
    2. 通过从颈部解剖组织暴露气管,并做一个微小的切口进入气管,允许18 G灌洗导管通过它(图5)。
    3. 将BALF导管插入小鼠气管并重复注射0.5ml冰冷的PBS/5%FBS(来自填充有3ml PBS/5%FBS的"输入"注射器),然后从膨胀的肺中抽吸BALF进入最初未填充的"输出"注射器通过三通停止。通常,我们注射总共3毫升,并在输出注射器中回收2.5毫升BALF。在每次注射和抽吸步骤后调节三通旋塞阀,以确保BALF被捕获在输出注射器中。
    4. 将BALF转移到空管中并储存在冰上。
    5. 在含有2ml冰冷PBS的管中收集肺并储存在冰上。


      图5.使用3向旋塞阀的bALF收获

  4. 肺部治疗
    1. 将肺转移到温和的MACS C管中,每管含有5ml新鲜制备的肺消化缓冲液
    2. 将管连接到温和的MACS解离器和运行程序"m_lung_01",以解离肺样品。在37℃下在MACS混合管旋转器上孵育45分钟以允许消化。
    3. 将管回到温和的MACS分离器并运行程序"m_lung_02,这完成肺细胞样品的单细胞悬浮液的准备。
    4. 短暂旋转管(300×g,1分钟)以使细胞在底部,并使用移液管,将内容物转移到新鲜的15ml管中。
      注意:除非另有说明,否则所有后续步骤均应在4°C下进行。
    5. 离心管在300×g离心5分钟,丢弃上清液和裂解红细胞,通过在室温下在1ml的1×RBC裂解缓冲液中重悬沉淀5分钟。
    6. 在RBC裂解后,向每个管中加入9ml冰冷的5%FBS/PBS,并将整个10ml肺悬浮液通过100μm细胞过滤器进入新鲜的15ml管中。
    7. 离心管,弃去上清液并将细胞重悬于2ml冰冷的5%FBS/PBS中
    8. 在自动细胞计数器中计数细胞,在U底96孔板中转移200μl细胞悬浮液(约2-5百万细胞),并准备用荧光抗体染色。

  5. BALF处理
    1. 含有BALF的旋管在300×g下离心5分钟,弃去上清液,并将沉淀转移到U形底96孔板中,用荧光抗体染色。
  6. 肺和BALF细胞悬浮液的流式细胞术
    1. 重悬肺和BALF样品在50微升的FACS缓冲液含有1:100稀释的Fc块,在4℃孵育10分钟。
    2. 在FACS缓冲液中洗涤细胞一次,加入50μl含有以下所列抗体(购自ebiosciences/BD/AbD serotec)的染色混合物混合物:
      1. Ab染色混合物用于肺嗜中性粒细胞和Ly6C高单核细胞(图6) CD45.2(克隆104)-CPCP-Cy5.5
        Ly6G(1A8) - FITC
        Ly6C(克隆AL-21)-PE-Cy7
        Ly6B.2(克隆7/4)-AF700
        CD11b(克隆M1/70) - 太平洋蓝
        中性粒细胞:CD45.2 + Ly6G + Ly6C CD11b + Ly6B.2 >
        单核细胞:CD45.2 + Ly6G
        Ly6C CD11b + Ly6B.2 >


        图6.用于测量肺组织中Ly6C高级单核细胞和嗜中性粒细胞的分生孢子吸收和杀伤的门控策略。在右栏中,红门由具有活分生孢子的白细胞,具有死分生孢子的蓝色门白细胞和不含分生孢子的黑门旁白细胞。

      2. Ab染色混合物用于肺巨噬细胞和CD11b + DC(图7) CD45.2(克隆104)-CPCP-Cy5.5
        CD103(2E7) - FITC
        CD11c(HL3)-PE-Cy7
        MHC II类(M5/114.15.2) - AF700
        CD11b(克隆M1/70) - 太平洋蓝
        巨噬细胞:CD45.2 + CD11c + MHCII var 自身荧光细胞
        CD11b + DCs:CD45.2 + CD11c + MHCII var CD103 - +


        图7.通过CD11b + DC和肺组织中的巨噬细胞测量分生孢子吸收和杀伤的门控策略在右栏中,红门由具有活分生孢子的白细胞,具有死分生孢子的蓝色门白细胞和不含分生孢子的黑门旁白细胞。

      3. Ab染色混合物用于BALF嗜中性粒细胞和肺泡巨噬细胞(图8) CD45.2(克隆104)-CPCP-Cy5.5
        Ly6G(1A8) - FITC
        CD11c(HL3)-PE-Cy7
        Ly6B.2(克隆7/4)-AF700
        CD11b(克隆M1/70) - 太平洋蓝
        中性粒细胞:CD45.2 + Ly6G + CD11c -CD11b + Ly6B.2 >
        肺泡巨噬细胞:CD45.2 + Ly6G CD11b Ly6B.2 -CD11c sup>
        注意:在所有FLARE实验中保持AF633和DsRed通道打开。白细胞是AF633 + DsRed + 具有活分生孢子,AF633 + DsRed - 的细胞具有死亡分生孢子,白细胞是AF633 - DsRed - 代表旁观者群体(Jhingran等,2012)。


        图8.用于测量BALF嗜中性粒细胞和肺泡巨噬细胞的分生孢子吸收和杀死的门控策略在右栏中,红门由具有活分生孢子的白细胞,具有死细胞的蓝门细胞白细胞分生孢子和不含分生孢子的黑门旁观白细胞
    3. 在暗处在4℃孵育20分钟。 洗涤一次,重悬在FACS缓冲液中,并在1小时内用流式细胞术分析。 我们不建议固定样品,因为我们观察到DsRed荧光强度显着降低,如果样品用1%PFA固定和分析24小时后。
      推荐的带通滤波器设置:
      DsRed(PE):586/15
      PerCP-Cy5.5:695/40
      FITC:530/30
      PE-Cy7:780/60
      AF700:720/40
      太平洋蓝:450/50
      AF633(APC):660/20

食谱

  1. PBS Tween(0.025%)
    0.025%吐温20(v/v,2.5ml,来自10%储液) 1,000ml PBS(pH7.4)
    过滤灭菌并在4℃下保存
  2. 三氯甲烷(1M,pH 8)
    121.14克三碱值
    溶于800ml Milli Q水中
    用10N HCl调节pH至8.0 将体积调节至1,000 ml,并在4°C下保存
  3. 碳酸氢钠缓冲液(0.1M,pH 9.5)
    8.40g NaHCO 3水溶液 3.56g Na 2 CO 3 sub。 溶解在1000ml Milli Q水中,并在4℃下保存
  4. 肺消化缓冲液(每只肺)
    5ml 5%FBS/PBS(pH7.4) 2.2mg/ml IV型胶原酶
    100μg/ml DNase I
  5. FACS缓冲区
    0.5%BSA(2.5g) 0.1mM EDTA(100μl,来自0.5M储备液) 500ml 1×PBS(pH7.4) 储存于4°C

致谢

这些研究在以下资助机构和拨款的支持下进行:Lucille Castori中心 微生物,炎症和癌症(CMIC)博士后联合到A.J.,NIH授予R01 AI093808和R21 AI105617到T.M.H。 T.M.H.是由Burroughs Wellcome基金支持的传染病发病机制中的研究者。这项研究部分通过NIH/NCI癌症中心支持资助P30 CA008748资助。

参考文献

  1. Espinosa,V.,Jhingran,A.,Dutta,O.,Kasahara,S.,Donnelly,R.,Du,P.,Rosenfeld,J.,Leiner,I.,Chen,CC,Ron,Y.,Hohl ,TM和Rivera,A。(2014)。  炎症单核细胞在肺中协调先天性抗真菌免疫。 PLoS Pathog 10(2):e1003940。
  2. Hasenberg,M.,Kohler,A.,Bonifatius,S.,Jeron,A.and Gunzer,M.(2011)。  使用双光子显微镜直接观察受感染肺中嗜中性粒细胞的吞噬作用和NET形成。 J Vis Exp (52)。
  3. Heung,L.J.,Jhingran,A.和Hohl,T.M。(2015)。 

    参考文献

    1. Espinosa,V.,Jhingran,A.,Dutta,O.,Kasahara,S.,Donnelly,R.,Du,P.,Rosenfeld,J.,Leiner,I.,Chen,CC,Ron,Y.,Hohl ,TM和Rivera,A。(2014)。  炎症单核细胞在肺中协调先天性抗真菌免疫。 PLoS Pathog 10(2):e1003940。
    2. Hasenberg,M.,Kohler,A.,Bonifatius,S.,Jeron,A.and Gunzer,M.(2011)。  使用双光子显微镜直接观察受感染肺中嗜中性粒细胞的吞噬作用和NET形成。 J Vis Exp (52)。
    3. Heung,L.J.,Jhingran,A.和Hohl,T.M。(2015)。 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jhingran, A., Kasahara, S. and Hohl, T. M. (2016). Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia . Bio-protocol 6(18): e1927. DOI: 10.21769/BioProtoc.1927.
  2. Heung, L. J., Jhingran, A. and Hohl, T. M. (2015). Deploying FLAREs to visualize functional outcomes of host-pathogen encounters. PLoS Pathog 11(7): e1004912.
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