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Jun 2020
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Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay
豌豆蚜虫饲养、细菌感染及血细胞吞噬功能测定   

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Abstract

Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects’ innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable method for small insects hemocyte phagocytosis assay by taking pea aphid as an example. Furthermore, we provide the protocols for pea aphid rearing and bacterial infection, which offer referential method for related research.

Keywords: Immune (免疫的), Protocol (方案), Phagocytosis (吞噬作用), Aphid rearing (蚜虫饲养), Bacterial infection (细菌感染)

Background

Phagocytosis, defined as the cellular uptake of particles bigger than 0.5 μm through formation of a membrane derived phagosome, is an ancient and evolutionarily conserved mechanism of insects cellular response (Lemaitre and Hoffmann, 2007; Melcarne et al., 2019a and 2019b). Phagocytosis is mediated by phagocytes, the dedicated cells, which can digest both “altered-self” particles and pathogens (Hillyer and Strand, 2014; Melcarne et al., 2019b). The phagocytes can be not only circulating hemocytes in hemocoel but also sessile hemocytes on tissues (Hillyer and Strand, 2014; Hillyer, 2016; Sigle and Hillyer, 2016). When pathogens enter hemocoel of insects, the phagocytes rapidly phagocytose pathogens and phagocytosis generally finishes in hours (Hillyer et al., 2003; King and Hillyer, 2012; Sigle and Hillyer, 2016).

There are mainly four different methods to perform hemocyte phagocytosis assay:
In vivo phagocytosis: (1) The insects are injected with fluorescently labeled latex beads or fluorescein-labeled dead bacteria. After incubation, trypan blue is injected to quench extracellular fluorescence. Then, the fluorescence is detected on a fluorescence microscope and the fluorescence intensity from dorsal vessel-associated hemocytes is quantified. The detailed methods were described in literature (Elrod-Erickson et al., 2000; Gonzalez et al., 2013; Garg and Wu, 2014; Nazario-Toole et al., 2018). For some insects, the fluorescent background in the hemolymph and tissues are strong and this may influence the results. (2) The insects are injected with fluorescently labeled latex beads or fluorescein-labeled bacteria. After incubation, the hemocytes are collected by ripping the larval in PBS solution containing trypan blue to quench extracellular fluorescence. The hemocytes are transferred and attached to a glass slide, and then the cells are fixed with formaldehyde. The phagocytosis is observed under microscope and the fluorescence intensity is quantified. The details were described previously (Kocks et al., 2005; Hao et al., 2018). For some insects, such as pea aphid, when inject bacteria into the body cavity, a lot of bacteria are centralized around the wound. Therefore, in vivo phagocytosis is not suitable for these insects.

Ex vivo phagocytosis: (1) After collected in insect medium in low binding tubes, hemocytes are mixed with fluorescein-labeled bacteria. Samples are incubated to enable phagocytosis, and then placed on ice to stop the process. Phagocytosis is quantified using a flow cytometer after quenching fluorescence of extracellular particles. The detailed method is described as previously (Melcarne et al., 2019b). (2) Drops of hemolymph are collected into insect medium and then mixed well with fluorescein-labeled bacteria. The samples are transferred to tissue culture round coverslips in a cell culture plate. After settled and adhered to the coverslips, the cells are fixed with paraformaldehyde. Then, F-actin of cells is stained with Phalloidin after permeabilized with Triton-100. Phagocytosis is observed under a laser scanning confocal microscope. The details of hemolymph collection and phagocytosis methods were described before (Schmitz et al., 2012; Ma et al., 2020).

The second method of ex vivo phagocytosis overcomes the difficulty due to small size of some insects for clean hemolymph collection. An accurate assay was described as previously: the fluorescence intensity in phagocytosing hemocytes is calculated and phagocytic index is represented as the capacity of hemocytes (Melcarne et al., 2019b). Combining these advantages, we provide a practicable method for pea aphid hemocyte phagocytosis assay in this study. Meanwhile, we provide protocols for pea aphid rearing and bacterial infection, which offer referential methods for related research.

Materials and Reagents

  1. Test tubes (Kangsheng Medical Glass Factory, Kangsheng, catalog number: GT-16125), diameter x length: 16 mm x 125 mm; Volume: 18 ml

  2. Conical flasks (Sichuan Glass Factory, Fuhai, catalog number: ZXP-250), volume: 250 ml

  3. Soil matrix (Shandong Luhao Agricultural Science and Technology co., Itd, Luhao), volume: 50 L

  4. Seedling pots, diameter: 13 cm (purchased from a local supermarket)

  5. Capillaries (Drummond, catalog number: 3-000-203-G/X)

  6. Centrifuge tubes 50 ml (KIRGEN, catalog number: KG2821); 1.5 ml (Axygen, catalog number: MCT-150-C)

  7. Sterile Petri dishes (Biofound, catalog number: FM-90-G)

  8. 48-well cell culture plates (Corning, Costar, catalog number: 3548)

  9. Tissue culture-treated round coverslips (8 mm diameter) (Solarbio, catalog number: YA0353-100)

  10. Broad bean (Vicia faba) seeds (purchased from local farmers market)

  11. A. pisum strain (originally collected from Yunnan, China and maintained in the lab of Prof. Zhiqiang Lu at Northwest A&F University)

  12. Gram-negative bacteria Pseudomonas aeruginosa (PAO1, from Dr. Xihui Shen at Northwest A&F University); Gram-positive bacteria Micrococcus luteus (Kept in the lab of Prof. Zhiqiang Lu at Northwest A&F University)

  13. NaCl (Guangdong Guanghua Sci-tech Co., Ltd, Huada, catalog number: 1.01307.040)

  14. Yeast extract (Oxoid, catalog number: LP0021)

  15. Tryptone (Oxoid, catalog number: LP0042)

  16. Phosphate-Buffered Saline (PBS) (Thermo Fisher, catalog number: 10010023)

  17. Agar power (Solarbio, catalog number: 9002-18-0)

  18. Grace’s medium (Sigma-Aldrich, catalog number: S0146)

  19. Phenylthiourea (Sigma-Aldrich, catalog number: P7629)

  20. Heat-inactivated fetal bovine serum (Biological Industries, catalog number: 04-011-1A)

  21. Escherichia coli (K-12) and Staphylococcus aureus Alexa Fluor 594 BioParticles (Invitrogen, catalog numbers: E23370 and S23372)

  22. 4% paraformaldehyde (Solarbio, catalog number: P1110)

  23. SF488 Phalloidin (Solarbio, catalog number: CA1646)

  24. Anti-fading reagent (Solarbio, catalog number: S2100)

  25. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

  26. Luria-Bertani liquid medium (see Recipes)

  27. Luria-Bertani agar medium (see Recipes)

  28. 0.85% NaCl solution (see Recipes)

  29. Hemolymph collection medium (see Recipes)

Equipment

  1. Dissecting forceps (ideal-tek, catalog number: 5.SA)

  2. Growth chamber (Ningbo Jiangnan Instrument Factory, model: GXZ-500B)

  3. Ultra-cold storage freezer (Whirlpool Corporation, Sanyo, model: 09S338)

  4. Ice machine (Scotsman ice systems Co., Ltd., Scotsman, model: MF36)

  5. Laminar flow cabinet (Shiheng Instrument Equipment Co., LTD., Dinco, model: SW-CJ-2F)

  6. Incubator (Taicang Experimental Equipment Factory, Peiying, model: HZQ-F100)

  7. Eppendorf Biophotometer (Eppendorf, catalog number: 6133000044)

  8. Laser scanning confocal microscope (Olympus, model: FV3000)

Software

  1. ImageJ (NIH, Bethesda, MD, USA)

  2. GraphPad Prism 5.0 (GraphPad, Inc., La Jolla, CA, USA)

Procedure

  1. Pea aphid rearing (Figure 1)

    1. Put the broad bean seeds into water for imbibing absorption of water adequately for 2 days.

    2. Remove the seeds peels gently.

    3. Plant one peeled seed about 1 cm depth in a seedling pot filled with soil matrix.

    4. The planted seeds grow in the growth chamber at 21 ± 1 °C and 70 ± 5% relative humidity under a 16-h light (L): 8-h dark (D) photoperiod.

    5. Seedlings with more than six leaves can be used.

    6. Place ten adult aphids on each seedling through clamping the tentacles with tweezers and allow to produce offspring in the growth chamber.

    7. Remove the adult aphids from the seedlings two days later.

    8. Rear the nymphs on the seedlings for nine days until they reach wingless adults.

    9. Use these newly emerged aphid adults to do the following experiments.



      Figure 1. Pea aphid rearing. Put the broad bean seeds into water for imbibing absorption of water adequately for 2 days (A and B). Plant one peeled seed about 1 cm depth into a seedling pot filled with soil matrix and then the seed sprouts (C). Place adult aphids on each seedling through clamping the tentacles with tweezers (D and E) and allow them to produce offsprings (F). Remove the adult aphids from the seedlings two days later and the nymphs grow into wingless adults nine days later (G).


  2. Bacterial infection

    1. Prepare Luria-Bertani liquid medium, Luria-Bertani agar medium, 0.85% NaCl and sterilize these reagents in an autoclave.

    2. Streak culture the P. aeruginosa and M. luteus (keep in a ultra-cold storage freezer at -80 °C) on Luria-Bertani agar plates respectively in an incubator at 37 °C overnight.

    3. Pick a P. aeruginosa bacterial colony into a test tube containing 5 ml Luria-Bertani liquid medium and pick a M. luteus bacterial colony into a conical flask containing 100 ml Luria-Bertani liquid medium in a laminar flow cabinet.

    4. Culture the bacteria in a bed temperature incubator at 37 °C with the rotate speed at 220 r/min.

    5. Measure the absorbance of the culture at 600 nm on an Eppendorf biophotometer until the optical density reaches approximately 1 (Figure 2A).

    6. Harvest the cells by centrifugation at 8,000 x g for 10 min and resuspend the pellets in sterilized 0.85% NaCl solution and wash the bacteria three times with 0.85% NaCl solution through centrifugation and resuspension.

    7. Bring the P. aruginosa suspension to 2 x 109 colony formation units (CFU)/ml (Figure 2B) and M. luteus cell suspension to 2 x 1010 CFU/ml.

    8. Anesthetize the adult aphids on ice for ten minutes and prick through the abdominal wall into the hemocoel of the aphids about 0.5 mm deep with a capillary dipped in bacteria suspensions or sterilized 0.85% NaCl solution (Figures 2C and 2D).



      Figure 2. Bacterial infection. Culture P. aeruginosa in a test tube containing 5 ml Luria-Bertani liquid medium until the optical density at 600 nm reach approximately 1 (A). Harvest the P. aeruginosa cells by centrifugation and resuspend the cells to 2 x 109 (CFU)/ml (B). Prick through the abdominal wall into the hemocoel of the aphids about 0.5 mm deep with a capillary dipped in the P. aeruginosa suspension (C and D).


  3. Hemocyte phagocytosis assays

    1. Anesthetize the adult aphids in a sterile Petri dish on ice for 10 min.

    2. Remove one aphid legs gently with dissecting forceps and mixed the drops of hemolymph with a 5 μl Grace’s medium drop containing 1 μM phenylthiourea and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS).

    3. Collect the hemolymph from 20 aphids per test group as above and mix the collected hemolymph well with 2 μl of 1mg/ml E. coli or S. aureus Alexa Fluor 594 BioParticles.

    4. Perform the operations of 5-14 steps in the dark.

    5. Transfer the prepared samples to tissue culture-treated round coverslips (8 mm diameter) in a 48-well cell culture plate.

    6. Allow the hemocytes to settle and adhere through incubating at room temperature for 1 h.

    7. Wash the coverslips twice with 200 μl hemolymph collection medium.

    8. Fix the washed hemocytes for 10 min with 200 μl 4% paraformaldehyde in PBS.

    9. Wash the coverslips three times (10 min each) with 200 μl PBS.

    10. Permeabilize the hemocytes with 200 μl 0.1% Triton X-100 in PBS for 10 min and wash twice with 200 μl PBS (10 min each).

    11. Incubate the permeabilized hemocytes with 200 μl diluted SF488 Phalloidin by 1:200 in PBS for 1 h.

    12. Wash the coverslips with 200 μl PBS three times (10 min each).

    13. Mount the coverslips on slides using anti-fading reagent (3 μl per coverslip).

    14. Observe and take photos under a laser scanning confocal microscope (Figure 3). The excitation and acquisition wavelengths of the bacteria were respectively 590 nm and 617 nm. The excitation and acquistion wavelengths of Phalloidin were respectively 495 nm and 519 nm.



      Figure 3. The confocal images of hemocytes with F-actin stained by SF-488 Phalloidin (A), E. coli AlexaFluo 594 BioParticles (B) and the overlay of hemocytes and E. coli. Scale bars: 5 μm.


    15. Calculate the bacterial fluorescence intensities in phagocytosing hemocytes with ImageJ software (The calculation processes were shown as Figure 4).

    16. Take the phagocytic index (PI) as the phagocytic capacity of hemocytes according to a previous description ( Melcarne et al., 2019b).



      Figure 4. The calculation processes of the bacterial fluorescence intensities in phagocytosing hemocytes with ImageJ software. The image (the image is from Figure 3B) was loaded into ImageJ software (A). The phagocytizing bacteria were circled (B) and the bacteria outside of the hemocyte were cleared out (C). The image type was set as 8 bit and inverted (D). Adjusted the threshold and chose the “Default” options as “B&W” (black and white) (E). Got the analyzed result after set the measurements and the IntDen (integrated density) value was represented the fluorescence intensity (F).

Data analysis

The data analysis of hemocyte phagocytosis assay is referred to our recent article ( Ma et al., 2020). The intensity of excitation light and the magnification times were the same during all observation processes. The photos taken from about twenty different views per coverslip were used to calculate the bacterial fluorescence intensities in phagocytosing hemocytes (the calculation processes were shown as Figure 4).

    PI calculation: Fraction of hemocytes phagocytosing (f) = number of hemocytes in fluorescence positive gate/total number of hemocytes. Phagocytic index (PI) = [mean fluorescence intensity of hemocytes in fluorescence positive gate] x f. Every experiment was performed three independent biological repetitions. All PI data was plotted using GraphPad Prism 5.0. Student’s t-test was used to determine other statistical values, which were presented as the mean ± SEM.

Notes

  1. Remove the legs of pea aphids gently to prevent linking of other tissues.

  2. Put the coverslips in the center of the cell culture plates. Make sure all the collected hemolymph and bacteria mixed samples bestrew the coverslips, but don’t flow to the lacuna neighbor to the wall of cell culture plate.

Recipes

  1. Luria-Bertani liquid medium

    Dissolve 5 g NaCl, 5 g Yeast extract and 10 g Tryptone in 1 L ddH2O and mix well

    Sterilize the medium in an autoclave

  2. Luria-Bertani agar medium

    Dissolve 5 g NaCl, 5 g Yeast extract, 10 g Tryptone, 15 g Agar power in 1 L ddH2O and mix well

    Sterilize the medium in an autoclave

  3. 0.85% NaCl solution

    Dissolve 0.85 g NaCl in 100 ml ddH2O and mix well and sterilize the solution in an autoclave

  4. Hemolymph collection medium

    Grace’s medium containing 1 μM phenylthiourea and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS)

Acknowledgments

This protocol is adapted from the publication of Ma et al. (2020). Funding for the study was provided by the National Natural Science Foundation of China grants (31970467 and 31772530) and the Fundamental Research Funds for the Central Universities (Z1090219001).

Competing interests

The authors declare no conflicts of interest.

References

  1. Elrod-Erickson, M., Mishra, S. and Schneider, D. (2000). Interactions between the cellular and humoral immune responses in Drosophila. Current Biology 10(13): 781-784.
  2. Garg, A. and Wu, L. P. (2014). Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus. Cell Microbiol 16(2): 296-310.
  3. Gonzalez, E. A., Garg, A., Tang, J., Nazario-Toole, A. E. and Wu, L. P. (2013). A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster. Curr Biol 23(22): 2319-2324.
  4. Hao, Y., Yu, S., Luo, F. and Jin, L. H. (2018). Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila. Cell Commun Signal 16(1): 95.
  5. Hillyer, J. F., (2016). Insect immunology and hematopoiesis. Dev Comp Immunol 58: 102-118.
  6. Hillyer, J. F., Schmidt, S. L. and Christensen, B. M. (2003). Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J Parasitol 89(1): 62-69.
  7. Hillyer, J. F. and Strand, M. R. (2014). Mosquito hemocyte-mediated immune responses. Curr Opin Insect Sci 3: 14-21.
  8. King, J. G. and Hillyer, J.F. (2012). Infection-induced interaction between the mosquito circulatory and immune systems. PLoS Pathog 8(11): e1003058.
  9. Kocks, C., Cho, J. H., Nehme, N., Ulvila, J., Pearson, A. M., Meister, M., Strom, C., Conto, S. L., Hetru, C., Stuart, L. M., Stehle, T., Hoffmann, J. A., Reichhart, J. M., Ferrandon, D., Ramet, M. and Ezekowitz, R. A. (2005). Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123(2): 335-346.
  10. Lemaitre, B. and Hoffmann, J. (2007). The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697-743.
  11. Ma, L., Liu, L., Zhao, Y., Yang, L., Chen, C., Li, Z. and Lu, Z. (2020). JNK pathway plays a key role in the immune system of the pea aphid and is regulated by microRNA. PLoS Pathog 16(6): e1008627.
  12. Melcarne, C., Lemaitre, B. and Kurant, E. (2019). Phagocytosis in Drosophila: From molecules and cellular machinery to physiology. Insect Biochem Mol Biol 109: 1-12.
  13. Melcarne, C., Ramond, E., Dudzic, J., Bretscher, A. J., Kurucz, E., Ando, I. and Lemaitre, B. (2019). Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster. FEBS J 286(14): 2670-2691.
  14. Nazario-Toole, A. E., Robalino, J., Okrah, K., Corrada-Bravo, H., Mount, S. M. and Wu, L. P. (2018). The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster. J Immunol 201(4): 1154-1164.
  15. Schmitz, A., Anselme, C., Ravallec, M., Rebuf, C., Simon, J. C., Gatti, J. L. and Poirie, M. (2012). The cellular immune response of the pea aphid to foreigen intrusion and symbiotic challenge. PLoS One 7(7): e42114.
  16. Sigle, L. T. and Hillyer, J. F. (2016). Mosquito hemocytes preferentially aggregate and phagocytose pathogens in the periostial regions of the heart that experience the most hemolymph flow. Dev Comp Immunol 55: 90-101.

简介

[摘要]昆虫依靠简单但有效的先天免疫系统来抵抗感染。细胞和体液反应在昆虫的先天免疫系统中是相互联系和协同的。吞噬作用是一种主要的细胞反应。很难从豌豆蚜虫之类的小昆虫身上收集干净的血淋巴。在此,我们以豌豆蚜为例,为小昆虫血细胞吞噬作用测定提供了一种可行的方法。此外,我们提供了豌豆蚜虫饲养和细菌感染的协议我上,这提供了相关的研究参考方法。

关键词:免疫,方案,吞噬作用,蚜虫饲养,细菌感染



[背景技术] 吞噬作用,定义为颗粒大于0.5的细胞摄取微米通过形成膜衍生的吞噬体的,是一种古老的和进化上保守的机制昆虫细胞应答(勒梅特和霍夫曼,2007; Melcarne 。等人,2019a和2019 b)。吞噬作用是由吞噬细胞(专用细胞)介导的,吞噬细胞可以消化“改变自身”的颗粒和病原体(Hillyer和Strand,2014;Melcarne等,2019b)。吞噬细胞不仅可以是血细胞中的循环血细胞,而且可以是组织上的无柄血细胞(Hillyer和Strand,2014; Hillyer,2016; Sigle和Hillyer,2016)。当病原体进入昆虫血腔,吞噬细胞吞噬迅速病理克ENS和吞噬作用通常涂饰在小时(Hillyer等人,2003;王和Hillyer,2012;和事务所Hillyer,2016)。

有m个AIN LY 4二种fferent方法来执行血细胞的吞噬作用测定:

体内吞噬作用:(1)向昆虫注射荧光标记的乳胶珠或荧光素标记的死亡细菌。孵育后,注入锥虫蓝以淬灭细胞外荧光。然后,在荧光显微镜和背血管相关血细胞的荧光强度被量化。在文献中描述了详细的方法(Elrod-Erick son等人,2000; Gonzalez等人,2013; G arg和Wu,2014; Nazario-Toole等人,2018)。对于某些昆虫,血淋巴和组织中的荧光背景很强,这可能会影响结果。(2)给昆虫注射荧光标记的乳胶珠或荧光素标记的细菌。温育后,血细胞通过翻录在PBS溶液c中的幼虫收集ø ntaining台盼蓝以淬灭细胞外荧光。将血细胞转移并附着在载玻片上,然后用甲醛固定细胞。在显微镜下观察吞噬作用并定量荧光强度。的细节进行了先前描述的(科克小号等人,2005;浩等人,2018)。对于某些昆虫,如蚜虫豌豆,当注入细菌进入体腔,大量的细菌被集中腌肉Ñ d是伤口。因此,体内吞噬作用不适用于这些昆虫。

离体吞噬作用:(1)在低结合管的昆虫培养基中收集血细胞后,将血细胞与荧光素标记的细菌混合。温育样品以使其能被吞噬,然后将其置于冰上以终止该过程。在淬灭细胞外颗粒的荧光后,使用流式细胞仪对吞噬作用进行定量。详细方法如前所述(Melcarne et al。,2019b)。(2)滴的血淋巴被收集到昆虫培养基,然后用充分混合˚F luorescein标记的细菌。将样品转移至细胞培养板中的组织培养圆盖玻片上。沉淀并粘附在盖玻片上后,将细胞用多聚甲醛固定。然后,在用Triton-100透化后,将细胞的F-肌动蛋白用鬼笔环染色。在激光扫描共聚焦显微镜下观察吞噬作用。之前已经描述了血淋巴收集和吞噬方法的详细信息(Schmitz等,2012;Ma等,2020)。

第二种离体吞噬作用的方法克服了由于一些昆虫的小尺寸而收集干净的淋巴的困难。之前描述了一种准确的测定方法:计算吞噬血细胞的荧光强度,并以吞噬指数表示血细胞的容量(Melcarne等,2019b)。结合这些优点,我们为这项研究中的豌豆蚜血细胞吞噬作用测定提供了一种可行的方法。同时,我们提供了豌豆蚜虫饲养和细菌感染的方案,为相关研究提供了参考方法。

关键字:免疫的, 方案, 吞噬作用, 蚜虫饲养, 细菌感染

 
材料和试剂
 
1.试管(抗生医学玻璃厂,抗生,Ç atalog号:GT-16125),d iameter X升ength:16毫米×125毫米; 容量:18毫升      
2.锥形瓶(蜀玻璃厂,富海,Ç atalog号:ZXP-250 )中,v olume:250毫升      
3.土壤基质(山东Luhao农业科技有限公司。ITD ,Luhao ),v olume:50升      
4.幼苗锅小号,直径:13厘米(从当地超市购买)      
5.毛细管(德拉蒙德,目录号:3-000-203-G / X)      
6.离心管小号50毫升(KIRGEN,目录号:KG2821); 1.5 ml(Axygen ,目录号:MCT-150-C)      
7.无菌培养皿ES (Biofound ,目录号: FM-90-G)      
8. 48孔细胞培养板小号(康宁,Costar公司,目录号:3548)      
9.经组织培养处理的圆形盖玻片(直径8毫米)(Solarbio ,目录号:YA0353-100)      
10.蚕豆(蚕豆蚕豆),种子(从当地农贸市场购买)   
11. A.蚜株(最初是从云南,中国收集和保存在教授的实验室志强卢在西北农林科技大学)   
12.革兰氏阴性菌铜绿假单胞菌(PAO1,来自西北农林科技大学的沉喜辉博士);革兰氏阳性菌微球菌(在不停教授的实验室志强卢在西北农林科技大学)   
13.氯化钠(广东光华科技Ç O,。大号TD,华达,Ç atalog号:1.01307.040)   
14.酵母提取物(Oxoid公司,Ç atalog号:LP0021)   
15.胰蛋白胨(Oxoid公司,Ç atalog号:LP0042)   
16.磷酸盐缓冲盐水(PBS)(赛默飞世,Ç atalog号:10010023)   
17.琼脂粉(Solarbio ,目录号:9002-18-0)   
18. Grace的介质(Sigma-Aldrich公司,Ç atalog号:S0146)   
19.苯基硫脲(Sigma-Aldrich公司,Ç atalog号:P7629)   
20.热灭活的胎牛血清(生物工业Ç atalog号:04-011-1A)   
21.大肠杆菌大肠杆菌(K-12)和金黄色葡萄球菌Alexa氟594生物颗粒(Invitrogen公司,ç atalog数小号:E23370和S23372)   
22. 4%多聚甲醛(Solarbio ,Ç atalog号:P1110)   
23. SF488鬼笔环肽(Solarbio ,Ç atalog号:CA1646)   
24.抗褪色试剂(Solarbio ,Ç atalog号:S2100)   
25.的Triton X-100(Sigma-Aldrich公司,Ç atalog号码:T8787)   
26. Luria-Bertani液体培养基(请参阅食谱)   
27. Luria-Bertani琼脂培养基(请参见食谱)   
28. 0.85%NaCl溶液(请参阅食谱)   
29.血淋巴收集培养基(请参阅食谱)   
 
设备
 
d issecting钳子(ideal- TEK ,目录号:5.SA)
生长室(宁波江南仪器厂,型号:GXZ-500B)
超冷存储冷冻柜(惠而浦公司,S anyo ,型号:09S338)
制冰机(苏格兰斯科特斯曼制冰系统有限公司,型号:MF36)
层流柜(石横仪器Ë quipment Ç O,LTD,。Dinco ,型号:SW-CJ-2F)
我ncubator(太仓实验设备厂,培英,型号:HZQ-F100)
Eppendorf 生物光度计(Eppendorf,目录号:6133000044)
激光扫描共聚焦显微镜(奥林巴斯,模式l :FV3000)
 
软件
 
ImageJ (NIH,美国马里兰州贝塞斯达)
GraphPad Prism 5.0 (GraphPad,Inc.,拉霍亚,CA,美国)
 


程序
 
豌豆蚜重新aring (图1)
将蚕豆种子放入水中吸收2天的水分。
轻轻地除去种子皮。
在装有土壤基质的育苗盆中播种一粒约1厘米深的去皮种子。
种植的种子在生长室在21±1 °C和70±5%相对湿度下在16小时光照(L):8小时黑暗(D)光周期下。
小号eedlings超过六个叶可以使用。
通过用镊子夹住触角,在每个幼苗上放置十只成年蚜虫,并在生长室中产生后代。
两天后从幼苗中除去成年蚜虫。
如果幼虫在幼苗上饲养了9天,直到它们到达没有翅膀的成虫。
使用这些新近出现的蚜虫成虫进行以下实验。
 


图1.豌豆蚜虫饲养。将蚕豆种子放入水中以充分吸收水2天(A和B)。植物ø约1cm深度成幼苗锅填满土基质,然后将种子发芽NE去皮种子小号(C)。通过用镊子(D和E)夹住触角,将成年蚜虫放在每棵幼苗上,并使其产生后代s (F)。两天后从幼苗中除去成年蚜虫,九天后若虫长成无翅成虫(G)。
 
细菌感染
准备Luria-Bertani液体培养基,Luria-Bertani琼脂培养基,0.85%NaCl,并在高压釜中对这些试剂进行灭菌。
划线培养的绿脓杆菌和藤黄微球菌(保持在一个超低温冰箱,在-80 ℃下分别)在Luria-Bertani琼脂糖板在培养箱中在37 ℃下过夜。
将铜绿假单胞菌细菌菌落挑入装有5毫升Luria-Bertani液体培养基,在层流柜中将一个黄褐菌细菌菌落放入装有100毫升Luria-Bertani液体培养基的锥形瓶中。
在37 °C的床温培养箱中以220 r / min的转速培养细菌。
测量在600nm在Eppendorf培养物的吸光度BioPhotometer生物分光光度计直到光密度达到ES approxima tely 1 (图2A) 。
通过以8,000 xg离心10分钟收获细胞,然后将沉淀重悬在灭菌的0.85%NaCl溶液中,并通过离心和重悬用0.85%NaCl溶液洗涤细菌3次。
使P. aruginosa悬浮液以2×10 9集落形成单位(CFU)/ ml的(图2B)和藤黄微球菌细胞悬浮液以2×10 10 CFU / ml的。
在冰上或十分钟内麻醉成年蚜虫,并用蘸有细菌悬液或灭菌的0.85%NaCl溶液的毛细管将腹壁刺入约0.5毫米深的蚜虫的血小管中(图s 2C和2D)。
 


图2.细菌感染。在装有5 ml Luria-Bertani液体培养基的试管中培养铜绿假单胞菌,直到600 nm处的光密度达到大约1(A)。通过离心收获铜绿假单胞菌细胞,并将细胞重悬至2 x 10 9 (CFU)/ ml(B)。用浸入铜绿假单胞菌悬液的毛细管(C和D)穿过腹壁刺入约0.5毫米深的蚜虫的血小管。
 
血细胞吞噬作用测定
麻醉成年蚜虫在无菌P ETRI二SH在冰上10分钟。
与解剖镊子轻轻取出一个蚜虫腿和与混合血淋巴的液滴5微升含1个Grace的介质降μM苯基硫脲和10%(体积/体积)热灭活的胎牛血清(FBS) 。
收集来自20只血淋巴每测试组蚜虫如上述并用2混合收集血淋巴以及微升1mg / ml的的大肠杆菌或金黄色葡萄球菌Alexa氟594生物颗粒。
执行在5-14步骤操作的黑暗。
将准备好的样品转移到48孔细胞培养板中经组织培养处理的圆形盖玻片(直径8 mm)上。
通过在室温下孵育1小时,使血细胞沉降并粘附。
用200μl血液淋巴收集培养基清洗盖玻片两次。
固定血细胞洗涤10分钟,用200 μl的PBS中的4%多聚甲醛。
洗盖玻片三次(每次10分钟)用200微升PBS 。
透的血细胞用200微升0.1%TRIT上X -100的PBS 10分钟并洗涤TWIC e为200微升PBS(每次10分钟)。
孵育透血细胞用200微升稀释SF488鬼笔环肽按1:200在PBS中1 ħ 。
用200个洗盖玻片微升PBS三次(每次10分钟)。
使用抗褪色试剂(每个盖玻片3μl )将盖玻片安装在幻灯片上。
观察并采取激光扫描共聚焦microsco下照片PE (图3) 。细菌的激发和捕获波长分别为590 nm和617 nm。激发和习得的波长鬼笔环肽分别为495纳米和519纳米。
 


图3.用SF-488鬼笔环肽(A),大肠杆菌AlexaFluo 594生物颗粒(B)染色的F-肌动蛋白对血细胞的共聚焦图像,以及血细胞和大肠杆菌的覆盖物。比例尺:5μm 。
 
计算吞噬血细胞中细菌的荧光强度 使用ImageJ软件(计算过程如图4所示)。
根据先前的描述(Melcarne et al。,2019b),将吞噬指数(PI)作为血细胞的吞噬能力。
 


图4.用ImageJ软件计算吞噬血细胞中细菌荧光强度的过程。图像(图像来自图3 B)已加载到ImageJ软件(A)中。圈住吞噬细菌(B),清除血细胞外的细菌(C)。图像类型设置为8位并反转(D)。调整阈值并选择“默认”选项作为“黑白”(黑白)(E)。设置测量值后得到分析结果,IntDen (积分密度)值表示荧光强度(F)。
 
数据分析
 
血细胞ph值细胞增生测定的数据分析参考我们最近的文章(Ma等,2020)。在所有观察过程中,激发光的强度和放大倍数均相同。从每盖玻片约二十个不同的视图拍摄的照片被用来计算细菌在吞噬血细胞的荧光强度(计算过程被示出为图4 )。
PI计算:吞噬的血细胞分数(f )=荧光阳性门数中的血细胞数/血细胞总数。吞噬指数(PI)= [荧光阳性门中血细胞的平均荧光强度] x f 。每个实验被执行主编三个独立的生物学重复。所有PI数据W¯¯如用GraphPad 5.0绘制。学生牛逼-测试被用来确定其他统计值,分别表示为平均值±SEM。
 
笔记
 
轻轻移开豌豆蚜虫的腿,以防止其他组织的连接。
将盖玻片放在细胞培养板的中心。确保收集的所有淋巴液和细菌混合样品都盖上盖玻片,但不要流到细胞培养板壁附近的空隙。
 
菜谱
 
Luria-Bertani液体培养基
d issolve 5克的NaCl ,5g的酵母提取物和10g胰蛋白胨1大号的DDH 2 O和混合以及
在高压灭菌器中灭菌培养基
Luria-Bertani琼脂培养基
d issolve 5克的NaCl ,5g酵母提取物,10g胰蛋白胨,15g的琼脂的功率在1大号的DDH 2 O和混合以及
在高压灭菌器中灭菌培养基
0.85%NaCl溶液
D将0.85 g NaCl溶解在100 ml ddH 2 O中,并充分混合并在高压釜中灭菌
血淋巴收集培养基
Grace的培养基含有1μM苯硫脲和10%(vol / vol)热灭活的胎牛血清(FBS)
 
致谢
 
该协议改编自Ma等的出版物。(2020)。资金,用于研究W¯¯作为由国家提供中国的赠款自然科学基金(31970467和31772530)和基础研究基金中央高校(Z1090219001)。
 
利益争夺
 
作者宣称没有利益冲突。
 
参考文献
 
1. Elrod-Erickson,M.,Mishra,S.,Schneider,D.(2000)。果蝇细胞和体液免疫反应之间的相互作用。现代生物学10(13):781-784。      
2. Garg,A.和Wu,LP(2014)。果蝇Rab14介导吞噬金黄色葡萄球菌的免疫反应。细胞微生物学16(2):296-310。      
3.冈萨雷斯(EA),加格(A.),唐(J.),纳萨里奥·塔特(Nazario-Toole),AE和吴(LP)(2013)。果蝇中依赖谷氨酸的氧化还原系统是果蝇吞噬作用所不可或缺的。Curr Biol 23(22):2319-2324。      
4.昊,Y.,玉,S.,罗,F和金,LH(2018)。果蝇是果蝇循环血细胞分化和吞噬所需要的。小区通信信号16(1):95。      
5.希勒,JF(2016)。昆虫免疫学和造血作用。Dev Comp Immunol 58:102-118。      
6. Hillyer,JF,Schmidt,SL和Christensen,BM(2003)。埃及蚊的血细胞快速吞噬和抑制细菌和子疟原虫的黑色素。J Parasitol 89(1):62-69。      
7. Hillyer,JF和Strand,MR(2014)。蚊子血细胞介导的免疫反应。Curr Opin昆虫科学3:14-21。      
8. King,JG和Hillyer,JF(2012年)。蚊子的循环系统和免疫系统之间的感染诱导相互作用。PLoS Pathog 8(11):e1003058。      
9. Kocks ,C.,Cho,JH,Nehme,N.,Ulvila ,J.,Pearson,AM,Meister,M.,Strom,C.,Conto ,SL,Hetru ,C.,Stuart,LM,Stehle , T.,Hoffmann,JA,Reichhart ,JM,Ferrandon ,D.,Ramet,M。和Ezekowitz ,RA(2005)。Eater是一种介导果蝇细菌病原体吞噬作用的跨膜蛋白。细胞123(2):335-346。      
10. Lemaitre,B.和Hoffmann,J.(2007)。果蝇的宿主防御。免疫学年报(Annu Rev Immunol)25:697-743。   
11. Ma,L.,Liu,L.,Zhao,Y.,Yang,L.,Chen C.,Li,Z. and Lu,Z.(2020)。JNK途径在豌豆蚜虫的免疫系统中起关键作用,并受microRNA-184调控。PLoS Pathog 16(6):e1008627。   
12. Melcarne ,C.,勒梅特,B。和Kurant ,E。(2019a)。果蝇中的吞噬作用:从分子和细胞机制到生理学。昆虫生物化学分子生物学109:1-12。   
13. Melcarne ,C.,小子,E.,Dudzic ,J.,Bretscher ,AJ,Kurucz ,E.,安藤,I。和勒梅特,B。(2019 b )。二尼姆罗德受体,NimC1和食,协同有助于细菌吞噬果蝇果蝇。FEBS J 286(14):2670-2691。   
14.扎里奥-图勒,AE,Robalino ,J.,Okrah ,K.,Corrada -Bravo,H.,芒,SM和Wu,LP(2018)。剪接因子RNA结合Fox蛋白1介导果蝇果蝇的细胞免疫反应。免疫学杂志201(4):1154-1164。   
15. Schmitz,A.,Anselme ,C.,Ravallec ,M.,Rebuf ,C.,Simon,JC,Gatti ,JL和Poirie ,M.(2012)。豌豆蚜虫对异源入侵和共生挑战的细胞免疫反应。PLoS One 7(7):e42114。   
16. Sigle,LT和Hillyer,JF(2016)。蚊子的血细胞优先聚集并吞噬经历最多淋巴流量的心脏骨膜区。Dev Comp Immunol 55:90-101。
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引用:Ma, L., Liu, L. and Lu, Z. (2020). Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay. Bio-protocol 10(24): e3862. DOI: 10.21769/BioProtoc.3862.
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