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Macrophage Infection by Fungi
真菌引起的巨噬细胞感染   

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Abstract

Mammalian infection by dimorphic fungi occurs through the inhalation of asexual spores (conidia), which are phagocytosed by host pulmonary alveolar macrophages of the innate immune system. Once phagocytosed, fungal conidia germinate into the pathogenic cell type; unicellular yeast cells which divide by fission (Vanittanakom et al., 2006; Boyce et al., 2011). To investigate if mutation of a particular fungal gene affects macrophage phagocytosis or the production of yeast cells, a murine macrophage cell culture assay can be utilized. This protocol was developed for Penicillium marneffei but is applicable to most dimorphic fungi.

Materials and Reagents

  1. Lipopolysaccharide (LPS) from E. coli (Sigma-Aldrich, catalog number: L2630 )
  2. Flask (or petri dish) of confluent J77A murine macrophages (available from Sigma-Aldrich, catalog number: 91051511 )
  3. 10x Trypsin-EDTA solution (Life Technologies, Gibco®, catalog number: R-001-100 )
  4. 1 x 107/ml fungal conidia (suspended in 0.001% Tween 20) (harvested from a 10 day 25 °C agar plate)
  5. 1 mg/ml fluorescent brightener 28 (calcofluor) (Sigma-Aldrich, catalog number: F3543 )
  6. 70% ethanol (any supplier)
  7. NaCl (ChemSupply, catalog number: SA046 )
  8. KCl (ChemSupply, catalog number: PA054 )
  9. MgSO4 (ChemSupply, catalog number: MA048 )
  10. NaOH (ChemSupply, catalog number: SA178 )
  11. Na2HPO4 (Thermo Fisher Scientific/Ajax Finechem Pty, catalog number: A621 )
  12. KH2PO4 (Merck KGaA, product number: 1048729025 )
  13. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 26140 )
  14. Penicillin streptomycin solution (Sigma-Aldrich, catalog number: P0781 )
  15. L-glutamine (Sigma-Aldrich, catalog number: 59202C )
  16. Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
  17. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  18. PIPES (Sigma-Aldrich, catalog number: P6757 )
  19. EGTA (Sigma-Aldrich, catalog number: E4378 )
  20. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  21. 4% fixation solution (see Recipes)
  22. Phosphate buffered saline (PBS) (see Recipes)
  23. Complete DMEM (see Recipes)
  24. 0.001% Tween 20 (see Recipes)
  25. PME buffer (see Recipes)

Equipment

  1. Standard tabletop centrifuges
  2. Clyde-Apac BH2000 Series Biological safety cabinet class II
  3. Leica Microscope with a UV filter (Reichert-Jung)
  4. Refrigeration centrifuge with a swing bucket rotor (Beckman Coulter, model: TJ-6 )
  5. Cell culture incubator (37°C, 5% CO2)
  6. Bunsen burner
  7. Well sterile cell culture plate (Greiner Bio-One, catalog number: 657160 )
  8. Disposable, sterile 10ml pipettes (Corning Incorporated/Costar stripettes, manufacture number: 4488 )
  9. Sterile 10 ml centrifuge tubes (any supplier)
  10. 22 x 22 mm standard microscope coverslips (any supplier)
  11. 76 x 26 mm standard microscope slides (any supplier)
  12. Nail varnish (any supplier)
  13. Biological safety cabinet
  14. Haemocytometer
  15. Tweezers
  16. 25 cm2 small flask
  17. 75 cm2 big flask

Procedure

Note: All steps performed in the Biological Safety Cabinet. Use sterile materials and reagents and aseptic techniques. All incubation steps are at 37 °C, 5% CO2 in a cell culture incubator unless indicated.
Day 1

  1. In the biological safety cabinet, gently remove the culture media from the flask (or petri dish) of confluent J774 murine macrophages and discard.
  2. Gently wash the cells 1x with 10 ml PBS.
  3. Add 1x Trypsin-EDTA solution to detach the cells (1 ml for a 25 cm2 small flask, 2.5 mL for a 75 cm2 big flask).
  4. Incubate for 30-60 min in the cell culture incubator to detach the cells.
  5. Add complete DMEM media to stop the digestion (4 ml for small flask, 7 ml for big flask) and transfer the cells into a sterile 10 ml centrifuge tube.
  6. Pellet the cells by centrifugation for 8 min at 1,500-1,800 rpm.
  7. Pour off media and discard and resuspend the cell pellet in 1 ml complete DMEM media. Calculate the concentration of cells using dilutions in PBS and a haemocytometer (want to add 1 x 105 cells total).
  8. Holding with fine tweezers, dip a coverslip into 70% ethanol, dab off excess ethanol by placing edge of the coverslip on a tissue and pass through the flame of a Bunsen burner to sterilze. Place the sterile coverslip into the well of a 6 well sterile cell culture plate. Repeat, placing a coverslip per well.
  9. Add 2 ml of complete DMEM media to each well of the 6 well cell culture plate. Add 1 x 105 macrophages per well. Incubate in the cell culture incubator overnight (24 h).

    Day 2
  10. Add 0.1 µg/ml LPS (in total volume) to each well of the 6 well cell culture plate. Incubate overnight (24 h) in the cell culture incubator.

    Day 3
  11. Remove the media from wells with a 10 ml pipette and discard. Pipette 5 ml of PBS into each well to wash the cells, then remove and discard. Repeat 3x in total.
  12. Add 2 ml fresh complete DMEM media to each well. Add 1 x 106 fungal conidia (10 µl of 1 x 107 fungal conidia/ml suspension) to each of the five wells (can do 5 different strains per experiment) leaving one well as a minus infection control.
  13. Incubate 2 h in the cell culture incubator (to allow phagocytosis of conidia).
  14. Remove media and discard. Gently wash off unbound spores with 5 ml PBS.
  15. To assess phagocytosis, continue straight to DAY 4.
    To assess yeast cell production, add 2 ml of fresh complete DMEM media and incubate for 24 h in the cell culture incubator.

    Day 4
  16. Remove the PBS or media from the wells and discard. Add 2 ml of 4% fixation solution. Leave 30 min at room temperature.
  17. Remove the fixation solution and discard. Add 2 ml of PME to each well to wash.
  18. On a microscope slide, place 5 µl 0.001% Tween 20. Add 1 µl of 0.1 mg/ml calcofluor to the Tween and mix using a pipette tip.
  19. Using fine tweezers, remove the coverslip from the cell culture plate well and invert, macrophage side down, onto a microscope slide.
  20. Blot with a tissue and seal with nail vanish.
  21. View on a microscope under the UV filter.

Recipes

  1. PBS (1 L)
    NaCl                     8 g
    KCl                      0.2 g
    Na2HPO4                 1.44 g
    KH2PO4                0.24 g
    Make up to 1 L in distilled water. Autoclave sterile.
  2. Complete DMEM (100 ml)
    10 ml FBS (when this first arrives it must be heat-inactivated for 30 min in 56 °C waterbath).
    Penicillin streptomycin solution   1 ml
    200 mM L-glutamine                8 ml
    DMEM                                   85 ml
  3. 4% fixation solution (100 ml)
    4 g of paraformaldehyde in 100 ml of PME buffer
    Wear mask when preparing
  4. PME buffer (1 L)
    PIPES                      15.12 g
    EGTA                       9.51 g
    1 M stock of MgSO4    5 ml
    pH to 6.7 using NaOH (the chemicals will not dissolve until the correct pH is almost reached)
  5. 0.001% Tween (1 L)
    10 μl of Tween 20 suspended in 1 L of water

Acknowledgments

This work was funded by grants from the Australian Research Council, National Health and Medical Research Council of Australia and the Howard Hughes Medical Institute.

References

  1. Boyce, K. J., Schreider, L., Kirszenblat, L. and Andrianopoulos, A. (2011). The two-component histidine kinases DrkA and SlnA are required for in vivo growth in the human pathogen Penicillium marneffei. Mol Microbiol 82(5): 1164-1184.
  2. Vanittanakom, N., Cooper, C. R., Jr., Fisher, M. C. and Sirisanthana, T. (2006). Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects. Clin Microbiol Rev 19(1): 95-110.

简介

两形态真菌的哺乳动物感染通过吸入无性孢子(分生孢子)发生,所述无性孢子被先天免疫系统的宿主肺泡巨噬细胞吞噬。 一旦被吞噬,真菌分生孢子萌发成致病细胞类型; 通过裂变分裂的单细胞酵母细胞(Vanittanakom et al。,2006; Boyce et al。,2011)。 为了研究特定真菌基因的突变是否影响巨噬细胞吞噬作用或酵母细胞的产生,可以利用鼠巨噬细胞培养测定法。 该方案是为马尼埃青霉开发的,但适用于大多数二形真菌。

材料和试剂

  1. 来自E的脂多糖(LPS) 大肠杆菌(Sigma-Aldrich,目录号:L2630)
  2. 汇合的J77A鼠巨噬细胞(可得自Sigma-Aldrich,目录号:91051511)的烧瓶(或培养皿)
  3. 10x胰蛋白酶-EDTA溶液(Life Technologies,Gibco ,目录号:R-001-100)
  4. 1×10 7/ml/ml真菌分生孢子(悬浮于0.001%Tween 20中)(从10天25℃琼脂平板收获)
  5. 1mg/ml荧光增白剂28(calcofluor)(Sigma-Aldrich,目录号:F3543)
  6. 70%乙醇(任何供应商)
  7. NaCl(ChemSupply,目录号:SA046)
  8. KCl(ChemSupply,目录号:PA054)
  9. MgSO 4(ChemSupply,目录号:MA048)
  10. NaOH(ChemSupply,目录号:SA178)

  11. (Thermo Fisher Scientific/Ajax Finechem Pty,目录号:A621)。

  12. (Merck KGaA,产品编号:1048729025)< br />
  13. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:26140)
  14. 青霉素链霉素溶液(Sigma-Aldrich,目录号:P0781)
  15. L-谷氨酰胺(Sigma-Aldrich,目录号:59202C)
  16. Dulbecco's Modified Eagle's Medium(DMEM)(Sigma-Aldrich,目录号:D6546)
  17. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  18. PIPES(Sigma-Aldrich,目录号:P6757)
  19. EGTA(Sigma-Aldrich,目录号:E4378)
  20. 吐温20(Sigma-Aldrich,目录号:P1379)
  21. 4%固定溶液(见配方)
  22. 磷酸盐缓冲盐水(PBS)(见Recipes)
  23. 完成DMEM(参见配方)
  24. 0.001%Tween 20(参见配方)
  25. PME缓冲区(参见配方)

设备

  1. 标准台式离心机
  2. Clyde-Apac BH2000系列生物安全柜II类
  3. 带有UV过滤器(Reichert-Jung)的Leica显微镜
  4. 带有摆动转子的制冷离心机(Beckman Coulter,型号:TJ-6)
  5. 细胞培养孵育器(37℃,5%CO 2)
  6. 本生灶
  7. 孔无菌细胞培养板(Greiner Bio-One,目录号:657160)
  8. 一次性无菌10ml移液管(Corning Incorporated/Costar stripettes,制造号:4488)
  9. 无菌10ml离心管(任何供应商)
  10. 22 x 22毫米标准显微镜盖玻片(任何供应商)
  11. 76 x 26 mm标准显微镜载玻片(任何供应商)
  12. 指甲油(任何供应商)
  13. 生物安全柜
  14. 血细胞计数器
  15. 镊子
  16. 25cm 2小瓶
  17. 75cm 2 大瓶

程序

注意:在生物安全柜中执行的所有步骤。 使用无菌材料和试剂和无菌技术。 除非指明,否则所有孵育步骤均在37℃,5%CO 2下在细胞培养箱中进行。
第1天

  1. 在生物安全柜中,轻轻地从合并的J774鼠巨噬细胞的培养瓶(或培养皿)中取出培养基并丢弃。
  2. 用10 ml PBS轻轻洗涤细胞1次。
  3. 加入1×胰蛋白酶-EDTA溶液以分离细胞(对于25cm 2小烧瓶为1ml,对于75cm 2大瓶为2.5mL)。
  4. 在细胞培养箱中孵育30-60分钟以分离细胞
  5. 加入完全DMEM培养基以停止消化(小烧瓶为4ml,大烧瓶为7ml),并将细胞转移到无菌的10ml离心管中。
  6. 通过在1,500-1,800rpm离心8分钟来沉淀细胞
  7. 倒出培养基并丢弃并将细胞沉淀重悬在1ml完全DMEM培养基中。使用PBS中的稀释液和血细胞计数器计算细胞的浓度(总共加入1×10 5个细胞)。
  8. 拿着精细的镊子,将盖玻片浸入70%乙醇,通过将盖玻片的边缘放置在组织上,通过本生灯的火焰灭菌,通过多余的乙醇。将无菌盖玻片放入6孔无菌细胞培养板的孔中。重复,每孔放置盖玻片。
  9. 向6孔细胞培养板的每个孔中加入2ml完全DMEM培养基。 每孔加入1×10 5个巨噬细胞。 在细胞培养箱中孵育过夜(24小时)
    第2天
  10. 向6孔细胞培养板的每个孔中加入0.1μg/ml LPS(总体积)。 在细胞培养箱中孵育过夜(24小时)
    第3天
  11. 用10毫升移液器从孔中删除介质,并丢弃。 移取5ml PBS到每个孔中以洗涤细胞,然后移除并丢弃。 重复3x。
  12. 向每个孔中加入2ml新鲜的完全DMEM培养基。 向5个孔中的每一个加入1×10 6个真菌分生孢子(10μl的1×10 7个真菌分生孢子/ml悬浮液)(每个实验可以做5个不同的菌株 ),留下一个孔作为负感染对照。
  13. 在细胞培养箱中孵育2小时(以允许分生孢子的吞噬作用)
  14. 删除介质并丢弃。 用5ml PBS轻轻洗去未结合的孢子。
  15. 要评估吞噬作用,请继续直到第4天。
    为了评估酵母细胞生产,加入2ml新鲜的完全DMEM培养基,并在细胞培养箱中孵育24小时。

    第4天
  16. 从孔中取出PBS或培养基并丢弃。 加入2ml 4%固定液。 在室温下离开30分钟。
  17. 取出固定液并丢弃。 向每个孔中加入2ml PME以进行洗涤
  18. 在显微镜载玻片上,放置5微升0.001%吐温20.添加1微升0.1毫克/毫升calcofluor到吐温,并使用移液器吸头混合。
  19. 使用细镊子,从细胞培养板井盖和盖,倒置,巨噬细胞侧向下,到显微镜载玻片上。
  20. 污点与组织和密封与指甲消失。
  21. 在UV过滤器下的显微镜下观察。

食谱

  1. PBS(1 L)
    NaCl                    8克
    KCl                      0.2克
    Na 2 HPO 4                1.44 g
    KH 2 PO 4               0.24克
    用蒸馏水补足至1 L。 高压灭菌器无菌。
  2. 完成DMEM(100 ml)
    10ml FBS(当这第一次到达时,它必须在56℃水浴中热灭活30分钟)。
    青霉素链霉素溶液 1 ml
    200 mM L-谷氨酰胺               8 ml
    DMEM                                   85 ml
  3. 4%固定溶液(100ml) 4g多聚甲醛在100ml PME缓冲液中的溶液 准备
    时戴上口罩
  4. PME缓冲液(1 L)
    PIPES                      15.12克
    EGTA                       9.51克
    1M MgSO 4的储液   5 ml
    使用NaOH将pH调至6.7(化学品将不溶解,直到几乎达到正确的pH)
  5. 0.001%吐温(1L)
    将10μl悬浮于1L水中的吐温20

致谢

这项工作是由澳大利亚研究委员会,澳大利亚国家卫生和医学研究理事会和霍华德休斯医学研究所资助。

参考文献

  1. Boyce,K. J.,Schreider,L.,Kirszenblat,L.和Andrianopoulos,A。(2011)。 双组分组氨酸激酶 DrkA 和 SlnA em>是 需要用于在人类病原体中的 in vivo 生长。 Mol Microbiol 82(5):1164-1184。 />
  2. Vanittanakom,N.,Cooper,C.R.,Jr.,Fisher,M.C.and Sirisanthana,T。(2006)。 马尼埃青霉感染和流行病学和分子生物学方面的最新进展。 Clin Microbiol Rev 19(1):95-110。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Boyce, K. and Andrianopoulos, A. (2012). Macrophage Infection by Fungi. Bio-protocol 2(14): e236. DOI: 10.21769/BioProtoc.236.
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