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Isolation and Infection of Drosophila Primary Hemocytes
果蝇原代血细胞的分离和感染   

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Abstract

Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.

Keywords: Drosophila(果蝇), Hemocytes(血细胞), Infection(感染), Phagocytosis(吞噬), Phagosome maturation(吞噬体成熟)

Background

The experiment described below can be used to study phagosome biogenesis, maturation, and delivery to lysosomes. Bacterial accumulation has been well studied in the context of immuno-compromised Drosophila with defects in IMD or Toll signaling and the resulting reduced expression of antimicrobial peptides (e.g., Lemaitre and Hoffmann, 2007; Kleino and Silverman, 2014). Cellular responses to bacterial infections have been less investigated in Drosophila, with most studies focused on mutations that interfere with the initial phagocytic uptake of bacteria by hemocytes (Kocks et al., 2005; Parsons and Foley, 2016). Such bacterial uptake is straightforward to measure using FACS analysis (Tirouvanziam et al., 2004). For a detailed analysis of phagosomal maturation, however, we found it advantageous to examine individual hemocytes attached to a glass cover slip (Akbar et al., 2011; Rahman et al., 2012; Akbar et al., 2016) as this procedure offered us the best combination of temporal and spatial resolution for our studies of phagosome maturation.

Materials and Reagents

  1. Falcon tubes (15 ml) (Corning, Falcon®, catalog number: 352196 )
  2. Eppendorf tubes
  3. Cover glass, No 1.5, 22 mm2 (Corning, catalog number: 2850-22 )
  4. Petri dish (100 x 15 mm) (Corning, catalog number: 351029 )
  5. Kimwipe
  6. Micro slides (Corning, catalog number: 2948-75X25 )
  7. Sterile filter unit: 0.22 µm cellulose acetate filter flasks (Corning, catalog number: 430769 )
  8. Drosophila melanogaster wandering third instar larvae (Ashburner, 1989)
  9. E. coli (DH5α) constitutively expressing GFP
    1. peGFP (https://www.addgene.org/vector-database/2485/)
    2. pET-GFP-C11 (http://www.addgene.org/30183/)
  10. Bucket of ice
  11. LB growth media (Fisher Scientific, catalog number: BP1425-500 )
  12. Schneider’s Drosophila medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720 ) with 10% FBS (Atlanta Biologicals, Advantage, catalog number: S11095 )
  13. S2 cell media
  14. PBSS = 0.3% Saponin (Sigma-Aldrich, catalog number: S7900 ) in PBS
  15. 10% NGS
  16. Phalloidin Alexa 594 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A12381 )
  17. Vectashield (Vector Laboratories, catalog number: H-1000 )
  18. Clear nail polish
  19. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  20. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  21. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
  22. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
  23. Hydrochloric acid (HCl)
  24. Sodium hydroxide (NaOH)
  25. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 19208 )
  26. 10% normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 ) in PBSS
  27. 10x PBS (see Recipes)
  28. 8% paraformaldehyde (see Recipes)
  29. Fixative: 4% paraformaldehyde in PBS (see Recipes)

Equipment

  1. Spectrophotometer (Molecular Devices, model: SpectraMax M2 )
  2. Low speed centrifuge for 15 ml Falcon tubes (Eppendorf, model: 5804 R )
  3. Dissecting stereomicroscope with Leica L2 cold light source (Leica Microsystems, model: Leica L2 )
  4. Fine dissecting forceps (Fine Scientific Tools)
  5. 37 °C incubator with shaking (Eppendorf, New BrunswickTM, model: Innova® 44 )
  6. 25 °C incubator (BioCold Environment, model: BC49-IN )
  7. Dissecting dish
  8. Confocal Microscope
    Note: We use a Zeiss LSM510 (Zeiss, model: LSM510 ) with 63 x NA1.4 objective.

Software

  1. ImageJ (NIH)
  2. Prism (GraphPad)

Procedure

  1. Preparation of GFP-expressing E. coli
    1. Innoculate 50 ml LB broth with a GFP E. coli colony.
    2. Incubate overnight at 37 °C with shaking.
    3. Use spectrophotometer to measure optical density at 600 nm (OD600).
      1. Measure bacteria with broth, and broth alone.
      2. Subtract OD600 of fresh LB broth from bacteria with broth.
    4. Dilute bacteria to OD600 = 0.1
      1. Spin 10 ml of bacterial solution at 4,000 x g for 10 min in 15 ml Falcon tubes at room temperature.
      2. Decant LB broth.
      3. Re-suspend, by vortexing in sufficient 1x PBS, to adjust bacteria to an OD600 of 0.1.
      4. Aliquot 1 ml into Eppendorf tubes.
      5. Heat-kill by placing tubes at 65 °C for 20 min.
      6. Freeze aliquots and thaw when ready to use.
    5. Re-suspend bacteria in Schneider’s Drosophila medium with FBS
      1. Centrifuge Eppendorf tube with heat-killed GFP-bacteria at 4,000 x g for 10 min at room temperature.
      2. Decant PBS supernatant.
      3. Add 1 ml Schneider’s Drosophila medium and vortex to re-suspend.
      4. For the E. coli we use this yields about 40,000 bacteria per µl of solution.
      5. Once re-suspended, warm to 25 °C before exposure to cells.

  2. Primary hemocyte isolation
    1. Warm fresh Schneider’s Drosophila medium to 25 °C.
    2. Collect ten wandering third instar larvae in a large drop of water on dissecting dish–wash thoroughly and place in fresh drop.
    3. Place a 22 mm2 coverslip in a 100 x 15 mm Petri dish.
    4. Add 200 μl Schneider’s Drosophila medium from step B1 to the coverslip.
      1. This should create a large droplet.
      2. Keep the area of the droplet as small as possible.
      3. Do not let the droplet spread out across the coverslip.
    5. Place the washed larvae into the large droplet on the coverslip.
    6. Under a dissecting microscope, use forceps to pinch and immobilize the tail end of the larva and use another forceps to grab and rip the outer cuticle of the larvae from tail to mouth (see Figure 1).


      Figure 1. Larval dissection. This image depicts the way forceps are used to hold (left forceps) and dissect the cuticle of larvae covered with S2 cell media with the right forceps along the larval midline (jagged black line) thereby releasing the larval hemolymph into the S2 cell media.

    7. Rip open all ten larvae in the media droplet on the coverslip.
      Note: Do this step as quickly as possible.
    8. Remove larval carcasses from the media droplet.
    9. Incubate hemolymph in media on coverslip in the Petri dish to allow hemocytes to attach to coverslip (at 25 °C for 15 min).
      1. This 15-min incubation will be sufficient time for hemocytes to adhere to the coverslip.
      2. After about 10 min, melanization may begin to occur (see Figure 2).
      3. Begin washes with PBS if the media features small black spots. These indicate the beginning of melanization that we seek to avoid.
      4. Do not allow the media to turn black.


        Figure 2. Early signs of melanization. The image displays a coverslip (22 x 22 mm) with a drop of medium containing hemocytes settling on the coverslip. Over time, crystal cells in the medium will initiate melanization that can be detected by the dark spots in the solution (arrow) and the dark ring at the edge of the drop (arrowhead). At this stage, hemocytes should be washed with PBS to remove crystal cells.

    10. Pour 1x PBS carefully into the Petri dish away from the coverslip to avoid dislodging the adherent cells.
      1. Wash thoroughly by slowly swirling the dish.
      2. Pour off PBS from Petri dish.
      3. Repeat wash (B10).
    11. Hemocytes will adhere to the coverslip, while the PBS washes remove the majority of crystal cells, which are mainly responsible for melanization.
    12. Remove the coverslip with forceps and place on Kimwipe, then place in a dry Petri dish on ice.

  3. Expose hemocytes to GFP bacteria
    1. Add enough GFP bacteria in Schneider’s Drosophila medium from step A5 (40,000 bacteria/µl) to cover the entire coverslip without spilling over the edge of the coverslip (~200 µl).
    2. Incubate bacteria with hemocytes on ice for 20 min to allow the bacteria to adhere to the cell surface.
    3. Rinse 3 x by pouring PBS into the Petri dish and swirling gently. Remove PBS by pouring off.
    4. Remove coverslip with forceps and place on Kimwipe, then place in dry Petri dish.
    5. Add fresh, warm Schneider’s Drosophila medium on the coverslip for the desired amount of chase time (in wild-type hemocytes the majority of bacteria will be digested and undetectable by 45 min).

  4. Fix, stain, and mount hemocytes (all performed at room temperature)
    1. Wash the media with PBS as before.
    2. Add enough 4% paraformaldehyde to cover the coverslip without spilling over its edges.
    3. Fix cells for 30 min.
    4. Thoroughly wash cells with PBS as before.
    5. Add PBSS for 30 min to permeabilize cell membranes.
    6. Remove PBSS and add 10% NGS in PBSS for 30 min to block unspecific antibody binding.
    7. Stain with primary and secondary antibodies of your choice and/or Phalloidin-Alexa 594 in PBSS for one hour.
      Note: Keep specimen in the dark as much as possible during and after secondary antibody staining.
    8. Wash with PBS 3 x 15 min after each antibody.
    9. Place the coverslip on kimwipe to dry it.
    10. Add a small drop of Vectashield to a microscope slide.
    11. Slowly lay the coverslip with stained hemocytes onto the drop of Vectashield.
    12. Seal the edges of the coverslip to the slide with nail polish.
    13. Image the cells on a confocal microscope with a 63x objective and 3x digital zoom.

Data analysis

To determine the number of remaining bacteria at different time points in different genetic backgrounds, confocal images were opened with ImageJ (NIH) and smoothed with a Gaussian blur of one before merging bacteria and phalloidin-stained channels. The number of bacteria within individual cells was counted and recorded in a Prism (GraphPad) spreadsheet as a single data point for each cell. Bacteria were counted when the entire GFP bacterium was surrounded by phalloidin staining. At least 25 cells were counted for each of three experiments. Prism software was used to plot a box and whisker graph and to perform a one-way ANOVA comparing relevant data sets (all to all).

Notes

  1. The experiment described above has been beneficial to our lab’s analysis of bacterial accumulation and phagosome maturation within primary hemocytes (Figure 3) and the effect of different genotypes (Akbar et al., 2011; Rahman et al., 2012; Akbar et al., 2016). Chase time and antibody conditions can be adapted for a wide variety of experimental questions. However, great care must be taken during the initial steps of hemocyte isolation from larvae. As mentioned above, the Schneider’s Drosophila medium cell media droplet must maintain a dome-like shape in order to contain the larvae within the droplet and to keep hemocytes in a defined area with ample nutrients.


    Figure 3. Micrograph of hemocyte and GFP-expressing bacteria of various stages of phagocytosis. Hemocyte surface, phagosomes and phagolysosomes are highlighted by staining for Hook (red, Krämer and Phistry, 1996) and Spinster (blue, Sweeney and Davis, 2002).

  2. Hemocytes isolated by our procedure are mostly phagocytic active plasmatocytes, but also contain crystal cells that make up some 5% of the hemocyte pool (Tepass et al., 1994). Upon activation crystal cells secrete prophenoloxidase which is activated by proteolysis. Subsequently, the activated phenoloxidase initiates a melanization reaction that interferes with imaging of bacterial degradation products (Neyen et al., 2014). Therefore, after all the larvae have been ripped open and subsequently removed from the media, it is critically important to prevent the Schneider’s Drosophila medium from turning black. The melanization process will start around 10 min after the larval dissecting begins and will proceed with hemocytes cannibalizing other hemolymph cells (i.e., crystal cells). Other researchers (Tirouvanziam et al., 2004) added protease inhibitors to the isolation medium to suppress the protease cascade that initiates melanization. We found, however, that our approach was sufficient to minimize melanization, while avoiding any potential effect of the protease inhibitor on phagosome maturation and bacterial degradation.

Recipes

  1. 10x PBS stock solution
    80 g NaCl
    2 g KCl
    26.8 g Na2HPO4·7H2O
    2.4 g KH2PO4
    Dissolve in 800 ml ddH2O
    Adjust pH with HCl to 7.4
    Add ddH2O to 1 L and mix well
  2. 8% paraformaldhyde stock solution
    1. Heat 200 ml dH2O to 55 °C (but not warmer!)
    2. Add 2 drops 50% w/w NaOH
    3. Add 20 g paraformaldehyde
    4. Stir continuously until clear
    5. Vacuum filter through with 0.22 µm filter
    6. Add ddH2O to adjust final volume to 250 ml
    7. Aliquot 2 ml into 15 ml Falcon tubes
    8. store at -80 °C
  3. Fixative: 4% paraformaldehyde in PBS
    1. Thaw a 2 ml aliquot of 8% paraformaldehyde
    2. Add 1.6 ml dH2O
    3. Add 400 µl 10x PBS stock solution
    4. Mix well

Acknowledgments

The work herein was supported by NIH Grants EY010199, EY021922. This protocol has been adapted and modified from our previous published work (Akbar et al., 2011; Akbar et al., 2016). The authors of this work declare no conflicts of interest.

References

  1. Ashburner, M. (1989). Drosophila: A laboratory manual. Cold Spring Harbor Laboratory Press.
  2. Akbar, M. A., Mandraju, R., Tracy, C., Hu, W., Pasare, C. and Kramer, H. (2016). ARC syndrome-linked Vps33B protein is required for inflammatory endosomal maturation and signal termination. Immunity 45(2): 267-279.
  3. Akbar, M. A., Tracy, C., Kahr, W. H. and Kramer, H. (2011). The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila. J Cell Biol 192(3): 383-390.
  4. Kleino, A. and Silverman, N. (2014). The Drosophila IMD pathway in the activation of the humoral immune response. Dev Comp Immunol 42(1): 25-35.
  5. 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.
  6. Krämer, H., and Phistry, M. (1996). Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies. J Cell Biol 133(6): 1205-1215.
  7. Lemaitre, B. and Hoffmann, J. (2007). The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697-743.
  8. Neyen, C., Bretscher, A. J., Binggeli, O. and Lemaitre, B. (2014). Methods to study Drosophila immunity. Methods 68(1): 116-128.
  9. Parsons, B. and Foley, E. (2016). Cellular immune defenses of Drosophila melanogaster. Dev Comp Immunol 58: 95-101.
  10. Rahman, M., Haberman, A., Tracy, C., Ray, S. and Kramer, H. (2012). Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes. Traffic 13(12): 1680-1692.
  11. Sweeney, S.T., and Davis, G.W. (2002). Unrestricted synaptic growth in spinster-a late endosomal protein implicated in TGF-beta-mediated synaptic growth regulation. Neuron 36(3): 403-416.
  12. Tepass, U., Fessler, L. I., Aziz, A. and Hartenstein, V. (1994). Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development 120(7): 1829-1837.
  13. Tirouvanziam, R., Davidson, C. J., Lipsick, J. S. and Herzenberg, L. A. (2004). Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes. Proc Natl Acad Sci U S A 101(9): 2912-2917.

简介

入侵病原体的吞噬作用及其随后在溶酶体中的清除对于有机体适应性是重要的。我们设计了以下方案从野生型和突变型果蝇幼虫中提取吞噬性血细胞,并用GFP标记的E感染分离的血细胞。大肠杆菌以测量个体血细胞内吞噬和降解的速度。

背景 下面描述的实验可用于研究吞噬体的生物发生,成熟和向溶酶体的递送。细菌积累已经在免疫受损的果蝇的背景下得到充分的研究,其具有IMD或Toll信号传导中的缺陷,并导致抗微生物肽的表达降低(例如,Lemaitre和Hoffmann ,2007; Kleino和Silverman,2014)。细菌感染的细胞响应在果蝇中的研究较少,大多数研究集中于干扰血细胞对细菌的最初吞噬吞噬的突变(Kocks等人,2005年) ;帕森斯和福利,2016)。这种细菌摄取是直接使用FACS分析进行测量(Tirouvanziam等人,2004)。然而,对于吞噬体成熟的详细分析,我们发现检查附着在玻璃盖板上的个体血细胞是有利的(Akbar等人,2011; Rahman等人。 ,2012; Akbar等人,2016),因为这个过程为我们研究吞噬体成熟提供了时间和空间分辨率的最佳组合。

关键字:果蝇, 血细胞, 感染, 吞噬, 吞噬体成熟

材料和试剂

  1. Falcon管(15ml)(Corning,Falcon ®,目录号:352196)
  2. Eppendorf管
  3. 盖玻璃,1.5,22毫米<2>(康宁,目录号:2850-22)
  4. 培养皿(100 x 15毫米)(康宁,目录号:351029)
  5. Kimwipe
  6. 微型幻灯片(康宁,目录号:2948-75X25)
  7. 无菌过滤器:0.22μm醋酸纤维素过滤瓶(Corning,目录号:430769)
  8. 猕猴桃果蝇游荡三龄幼虫(Ashburner,1989)
  9. 电子。大肠杆菌(DH5α)组成型表达GFP
    1. peGFP( https://www.addgene.org/vector-database / 2485 /
    2. pET-GFP-C11()
  10. 冰桶
  11. LB生长培养基(Fisher Scientific,目录号:BP1425-500)
  12. 具有10%FBS(Atlanta Biologicals,Advantage,目录号:S11095)的施耐德果蝇培养基(Thermo Fisher Scientific,Gibco TM,目录号:21720)
  13. S2细胞培养基
  14. PBSS = 0.3%皂苷(Sigma-Aldrich,目录号:S7900)在PBS中
  15. 10%NGS
  16. Phalloidin Alexa 594(Thermo Fisher Scientific,Invitrogen TM,目录号:A12381)
  17. Vectashield(Vector Laboratories,目录号:H-1000)
  18. 透明指甲油
  19. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  20. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  21. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:S9390)
  22. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285)
  23. 盐酸(HCl)
  24. 氢氧化钠(NaOH)
  25. 多聚甲醛(Electron Microscopy Sciences,目录号:19208)
  26. 在PBSS中的10%正常山羊血清(Jackson ImmunoResearch,目录号:005-000-121)
  27. 10x PBS(参见食谱)
  28. 8%多聚甲醛(见配方)
  29. 固定剂:PBS中4%多聚甲醛(见配方)

设备

  1. 分光光度计(Molecular Devices,型号:SpectraMax M2)
  2. 用于15 ml Falcon管的低速离心机(Eppendorf型号:5804 R)
  3. 用Leica L2冷光源解析立体显微镜(Leica Microsystems,型号:Leica L2)
  4. 精细解剖钳(精细科学工具)
  5. 37°C振荡孵化器(Eppendorf,New Brunswick TM,型号:Innova ® 44)
  6. 25℃培养箱(BioCold Environment,型号:BC49-IN)
  7. 解剖盘
  8. 共焦显微镜
    注意:我们使用具有63 x NA1.4目标的Zeiss LSM510(Zeiss,型号:LSM510)。

软件

  1. ImageJ(NIH)
  2. 棱镜(GraphPad)

程序

  1. GFP表达的制备E。大肠杆菌
    1. 用绿色大肠杆菌菌落接种50ml LB肉汤。
    2. 在37℃下振荡孵育过夜。
    3. 使用分光光度计测量600nm处的光密度(OD 600)。
      1. 用肉汤和肉汤测量细菌。
      2. 用肉汤从细菌中减去新鲜LB肉汤的OD 600
    4. 将细菌稀释至OD 600 <0.1
      1. 在室温下,在15ml Falcon管中将10ml细菌溶液以4,000×g旋转10分钟。
      2. 滗水LB肉汤
      3. 通过在足够的1x PBS中涡旋重新悬浮,将细菌调节至0.1的OD 600。
      4. 将1ml等份装入Eppendorf管中
      5. 将管放置在65°C 20分钟即可杀死。
      6. 准备使用时冻结等分并解冻。
    5. 用FBS将细菌重新悬挂在施奈德的果蝇培养基中
      1. 将离心的Eppendorf管以4,000 x g的热灭活GFP细菌在室温下离心10分钟。
      2. 滗析PBS上清液
      3. 加入1毫升施奈德的果蝇培养基,涡旋重新悬挂。
      4. 对于大肠杆菌,我们使用这种方法每μl溶液产生约40,000个细菌。
      5. 一旦重新悬浮,在暴露于细胞前温热至25°C

  2. 原发性血细胞分离
    1. 温暖的新施耐德果汁中等至25°C。
    2. 收集十只流浪的三龄幼虫在一大滴水中彻底解剖清洗,放入新鲜的水滴。
    3. 将一个22毫米 2 盖玻片放在100 x 15毫米的培养皿中。
    4. 从步骤B1向盖玻片添加200μl施耐德果蝇培养基。
      1. 这应该会产生一个大的液滴
      2. 保持液滴的面积尽量小。
      3. 不要让液滴散布在盖玻片上。
    5. 将洗涤的幼虫置于盖玻片上的大液滴中。
    6. 在解剖显微镜下,使用镊子夹住并固定幼虫的尾端,并使用另一镊子从尾部到嘴部抓住并撕开幼体的外角质层(见图1)。


      图1.幼虫解剖。 该图像描绘了钳子用于保持(左镊子)的方式,并用右侧钳子沿着幼体中线(锯齿状黑线)剖开用S2细胞培养基覆盖的幼体的角质层,从而将幼虫血淋巴释放到S2细胞培养基
    7. Rip打开盖玻片上的媒体液滴中的所有十个幼虫。
      注意:尽快执行此步骤。
    8. 从媒体液滴中清除幼体尸体。
    9. 在培养皿中的盖玻片上的培养基中孵育血淋巴以允许血细胞附着于盖玻片(在25℃下15分钟)。
      1. 这种15分钟的孵化将足以使血细胞粘附到盖玻片上。
      2. 大约10分钟后,可能开始发生黑化现象(见图2)
      3. 如果媒体具有小的黑点,则开始使用PBS洗涤。这些表明我们试图避免的黑化开始。
      4. 不要让媒体变黑。


        图2.黑化的早期迹象。图像显示一个盖玻片(22 x 22毫米),一滴含有沉积在盖玻片上的血细胞的培养基。随着时间的推移,介质中的晶体细胞将引发黑色化,可以通过溶液中的黑点(箭头)和滴液边缘的黑色环(箭头)检测。在这个阶段,血细胞应该用PBS洗涤去除晶体细胞
    10. 将1x PBS小心地倒入盖玻片中的培养皿中,以避免剥离贴壁细胞。
      1. 慢慢地旋转盘子彻底清洗。
      2. 从培养皿中倒出PBS。
      3. 重复洗涤(B10)。
    11. 血细胞将粘附在盖玻片上,而PBS洗涤除去大多数主要负责黑化的晶体细胞。
    12. 用镊子取下盖玻片并放在Kimwipe上,然后放在冰上的干燥培养皿中。

  3. 将血细胞暴露于GFP细菌
    1. 在步骤A5(40,000细菌/μl)的施奈德果蝇培养基中添加足够的GFP细菌以覆盖整个盖玻片而不溢出盖玻片的边缘(〜200μl)。
    2. 将细菌与血细胞在冰上孵育20分钟,以使细菌粘附到细胞表面
    3. 将PBS倒入培养皿中冲洗3次,轻轻旋转。倾倒掉PBS。
    4. 用镊子取下盖玻片,放在Kimwipe上,然后放在干燥的培养皿中。
    5. 在盖玻片上添加新鲜温暖的施奈德果蝇培养基,达到所需的追逐时间(在野生型血细胞中,大部分细菌将被消化,45分钟不能检测到)。

  4. 修复,染色和挂载血细胞(均在室温下进行)
    1. 用PBS清洗培养基。
    2. 添加足够的4%多聚甲醛以覆盖盖玻片而不会溢出其边缘。
    3. 修复细胞30分钟。
    4. 用PBS彻底清洗细胞。
    5. 加入PBSS 30分钟以使细胞膜透化
    6. 去除PBSS并加入10%NGS在PBSS中30分钟以阻止非特异性抗体结合
    7. 用您选择的一级和二级抗体和/或Phalloidin-Alexa 594在PBSS中染色一小时。
      注意:在二次抗体染色期间和之后,尽可能多地将标本保持在黑暗中。
    8. 每次抗体后3×15分钟用PBS洗涤。
    9. 将盖玻片放在kimwipe上干燥。
    10. 在显微镜载玻片上添加一小滴Vectashield。
    11. 慢慢地将带有染色血细胞的盖玻片放在Vectashield的下落上
    12. 用指甲油将盖玻片的边缘密封到滑块上。
    13. 在共焦显微镜上用63x物镜和3倍数码变焦对图像进行成像。

数据分析

为了确定不同遗传背景下不同时间点的剩余细菌数,用ImageJ(NIH)打开共焦图像,并在合并细菌和鬼笔环素染色通道之前用高斯模糊平滑。计数单个细胞内的细菌数,并将其记录在Prism(GraphPad)电子表格中作为每个细胞的单一数据点。当整个GFP细菌被鬼笔环肽染色包围时,计数细菌。对于三个实验中的每一个计数至少25个细胞。 Prism软件用于绘制盒子和晶须图,并执行单因素方差分析比较相关数据集(全部)。

笔记

  1. 上述实验对我们实验室对原代血细胞内细菌积累和吞噬体成熟的分析(图3)和不同基因型的影响(Akbar等人,2011; Rahman et al。等等,2012年; Akbar等人,2016)。追踪时间和抗体条件可适用于各种实验问题。然而,在从幼体中分离血细胞的初始步骤中必须非常小心。如上所述,施耐德果蝇中等细胞培养基液滴必须保持穹顶状,以便容纳液滴内的幼虫,并将血细胞保持在具有丰富营养物质的限定区域内。


    图3.吞噬不同阶段的血细胞和GFP表达细胞的显微照片。通过染色Hook(红,Krämer和Phistry,1996)和Spinster(蓝色, Sweeney和Davis,2002)
  2. 通过我们的手术分离的血细胞主要是吞噬活性血浆细胞,但也含有构成血细胞池约5%的晶体细胞(Tepass等人,1994)。激活后,晶体细胞分泌由蛋白水解激活的酚氧化酶。随后,活化的酚氧化酶引发干扰细菌降解产物成像的黑化反应(Neyen等人,2014)。因此,在所有的幼虫被撕开并随后从培养基中除去之后,至关重要的是防止施耐德的果蝇介质变黑。黑化过程将在幼虫解剖开始后约10分钟开始,并且将进行血细胞吞噬其他血淋巴细胞(即,晶体细胞)。其他研究人员(Tirouvanziam等人,2004)将蛋白酶抑制剂加入到分离培养基中以抑制引发黑化的蛋白酶级联。然而,我们发现我们的方法足以减少黑化,同时避免蛋白酶抑制剂对吞噬体成熟和细菌降解的任何潜在影响。

食谱

  1. 10倍PBS储备溶液
    80克NaCl
    2克KCl
    26.8g Na 2 HPO 4&lt; 7H 2&lt; 2&gt;&lt; /&gt; 2.4g KH 2 PO 4
    溶于800毫升ddH 2 O - / - 用HCl调节pH至7.4
    将ddH 2 O添加到1L,并且很好地混合
  2. 8%多聚甲醛溶液
    1. 加热200毫升dH 2 O至55℃(但不加热!)
    2. 加入2滴50%w / w NaOH
    3. 加入20g多聚甲醛
    4. 持续搅拌直到清除
    5. 真空过滤器通过0.22μm过滤器
    6. 加入ddH 2 O以将最终体积调整至250ml
    7. 将等份2毫升装入15毫升Falcon管中
    8. 存放于-80°C
  3. 固定剂:PBS中4%多聚甲醛
    1. 解冻2毫升等份8%多聚甲醛
    2. 加入1.6ml dH 2 O O
    3. 加入400μl10倍PBS储备溶液
    4. 混合好

致谢

本文的工作由NIH Grants EY010199,EY021922支持。该协议已经从我们之前发表的作品(Akbar等人,2011; Akbar等人,2016)进行了修改和修改。这项工作的作者声明没有利益冲突。

参考

  1. Ashburner,M.(1989)。 果蝇:实验室手册冷泉港实验室出版社
  2. Akar,MA,Mandraju,R.,Tracy,C.,Hu,W.,Pasare,C.和Kramer,H。(2016)。 ARC综合征相关的Vps33B蛋白是炎症性内体成熟和信号终止所必需的。免疫性 45( 2):267-279。
  3. Akbar,MA,Tracy,C.,Kahr,WH和Kramer,H.(2011)。&nbsp; 在果蝇免疫防御期间,全细菌基因是吞噬细胞成熟所必需的。 J Cell Biol 192 3):383-390。
  4. Kleino,A.和Silverman,N。(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/23721820”target =“_ blank” >果蝇 IMD途径在体液免疫反应的激活中。 Dev Comp Immunol 42(1):25-35。
  5. Kocks,C.,Cho,JH,Nehme,N.,Ulvila,J.,Pearson,AM,Meister,M.,Strom,C.,Conto,SL,Hetru,C.,Stuart,LM,Stehle, ,Hoffmann,JA,Reichhart,JM,Ferrandon,D.,Ramet,M.and Ezekowitz,RA(2005)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm。 nih.gov/pubmed/16239149“target =”_ blank“>食者,介导果蝇中细菌病原体吞噬作用的跨膜蛋白。 123(2) :335-346。
  6. Krämer,H.和Phistry,M.(1996)。果蝇钩子基因中的突变抑制了上皮跨膜配体进入多泡体的内吞作用。 J Cell Biol 133(6):1205-1215。 br />
  7. Lemaitre,B.和Hoffmann,J.(2007)。果蝇黑腹果蝇的宿主防御 Annu Rev Immunol 25:697-743。
  8. Neyen,C.,Bretscher,AJ,Binggeli,O. and Lemaitre,B。(2014)。&nbsp; 研究免疫力的方法方法 68(1):116-128。
  9. Parsons,B.和Foley,E.(2016)。&nbsp; 果蝇 melanogaster的细胞免疫防御 Dev Comp Immunol 58:95-101。
  10. Rahman,M.,Haberman,A.,Tracy,C.,Ray,S。和Kramer,H。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/22934826“target =”_ blank“> 果蝇淡紫色突变体揭示了吞噬体和自噬体成熟后期的LYST同源物的作用。 / em> 13(12):1680-1692。
  11. Sweeney,S.T.和Davis,G.W。 (2002)。无限制的突触后增长在一个晚期涉及TGF-β介导的突触生长调节的内体蛋白。神经元 36(3):403-416。
  12. Tepass,U.,Fessler,LI,Aziz,A.and Hartenstein,V.(1994)。血液细胞的胚胎起源及其与果蝇中细胞死亡的关系 发展 120(7):1829-1837 。
  13. Tirouvanziam,R.,Davidson,CJ,Lipsick,JS和Herzenberg,LA(2004)。果蝇血细胞的荧光激活细胞分选(FACS)显示与哺乳动物白细胞重要的功能相似性。 Proc Natl Acad Sci USA 101(9):2912-2917。
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引用:Tracy, C. and Krämer, H. (2017). Isolation and Infection of Drosophila Primary Hemocytes. Bio-protocol 7(11): e2300. DOI: 10.21769/BioProtoc.2300.
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