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Drosophila Model of Leishmania amazonensis Infection
亚马逊利什曼原虫感染的果蝇模型   

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本实验方案简略版
PLOS Pathogens
Jun 2016

Abstract

This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.

Keywords: Drosophila melanogaster (黑腹果蝇), Leishmania (利什曼虫), Phagocytosis (吞噬作用), Plasmatocytes (浆细胞), Hemocytes (血细胞), Axenic amastigote (纯培养无鞭毛体), Amastigote (无鞭毛体), RNAi screen (RNAi 筛选)

Background

Leishmaniasis, caused by Leishmania protozoans, affects the skin, mucosa or internal organs, depending on the parasite species and immunological status of the host. Although the mouse model of infection has provided most of our understanding about this parasitic infection, it is not well suited for large-scale exploratory approaches. In our lab, we exploited the tractable and powerful genetics of the fruit fly Drosophila melanogaster to establish a model of Leishmania infection and perform a small-scale screen to identify host genes involved in the phagocytosis of these parasites (Okuda et al., 2016). In particular, a set of genes possibly linked to phagocytosis was specifically knocked-down in the fly hemocytes, using the UAS-GAL4 expression systems (Brand and Perrimon, 1993) and the survival and parasite burden of mutant-infected flies were monitored to identify factors modulating the infection.

Materials and Reagents

  1. 15 ml conical tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  2. Cell culture flasks (T-flask) 80 cm2 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178905 )
  3. T-flask 175 cm2 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
  4. Cell scraper (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 179707 )
  5. 10 ml serological pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 170356 )
  6. 1.5 ml microcentrifuge tube (Corning, Costar®, catalog number: 3622 )
  7. 96-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
  8. Pipette tips (Fisher Scientific, catalog numbers: 21-402-269 , 21-402-569 , 21-402-589 )
    Manufacturer: Thermo Fisher Scientific, catalog numbers: 3752RI , 3782RI .
  9. Disposable counting chamber (Bio-Rad Laboratories, catalog number: 1450003 )
  10. Pellet pestles (Fisher Scientific, catalog number: 12-141-364 )
  11. Fly vials (Genesee Scientific, Flystuff, catalog number: 32-116SB )
  12. Leishmania amazonensis (strain RAT/BA/LV78, Dutta et al., 2005)
  13. BALB/c bone marrow derived macrophages (BMDM)
    Note: Material and reagents are described in the Bio-protocol paper of Chen, 2013. Balb/c mice were obtained from The Jackson Laboratories. The femurs and tibias of one mouse generate approximately 3 x 108 macrophages.
  14. Phosphate-buffered saline (PBS) (Corning, catalog number: 21-031-CV )
  15. RPMI 1640 (Corning, catalog number: 10-040-CV )
  16. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  17. Bromophenol blue (Fisher Scientific, catalog number: BP115-25 )
  18. Fly food (FlyStuff Nutri-Fly Molasses Formulation or Nutri-FlyTM Bloomington Formulation, Genesee Scientific, Flystuff, catalog numbers: 66-116 )
  19. Yellow Cornmeal Fly food (Genesee Scientific, Flystuff, catalog number: 62-101 )
  20. Schneider’s insect medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720024 )
  21. Penicillin/streptomycin (Corning, catalog number: 30-002-Cl )
  22. Tetracyclin (Sigma-Aldrich, catalog number: T7660 )
  23. Tunicamycin (Cayman Chemical, catalog number: 11445 )
  24. 70% ethanol
  25. 199 media (Corning, catalog number: 90-050-PB )
  26. Heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S12450 )
  27. HEPES (Sigma-Aldrich, catalog number: H3784-100G )
  28. Sodium succinate (Sigma-Aldrich, catalog number: S9637 )
  29. Hemin (Sigma-Aldrich, catalog number: H9039 )
  30. Tryptic soy broth (BD, catalog number: 211825 )
  31. 199 media to grow promastigotes (see Recipes)
  32. 199 media for promastigote to axenic amastigote differentiation (see Recipes)

Equipment

  1. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM RT ), equipped with swinging buckets for 15 ml conical tubes
  2. Incubator (VWR® Water Jacketed CO2 Incubators)
  3. Glass dounce with grinding chamber and Teflon rod, clearance of 0.004” to 0.006” (DWK Life Sciences, Wheaton, catalog number: 358011 )
  4. Hemocytometer Hausser Scientific Bright-LineTM Counting Chamber (Hausser Scientific, catalog number: 3110 )
  5. Capillary puller (NARISHIGE, model: PC-100 )
  6. Nanoliter injector (Drummond Scientific, model: Nanoject II )
  7. Drummond Scientific Capillaries for Nanojet II (Pipette.com, catalog number: 3-000-203-G/X)
    Manufacturer: Drummond Scientific, catalog number: 3-000-203-G/X .
  8. Electronic caliper (Mitutoyo, catalog number: 500-197-30 )
  9. CO2 pad (Genesee Scientific, FlyStuff, catalog number: 59-114 )
  10. Stereomicroscope (Nikon Instruments, model: SMZ645 )
  11. Multichannel pipette (Thermo Fisher Scientific, Thermo ScientificTM, model: FinnpipetteTM F2 )
  12. Pellet grinder (Fisher Scientific, catalog number: 12-141-361 )
  13. Fine tweezers (Fisher Scientific, catalog number: 12-000-122 )
  14. Fluorescence microscope (Nikon Instruments, model: Eclipse E400 ) equipped with an imaging system (camera: Nikon Instruments, model: Ds-Qi1 , imaging software: NIS-Elements)

Software

  1. Imaging software: NIS-Elements Basic Reseach, Microscope Imaging Software (Nikon Instruments)
  2. GraphPad Prism7 (GraphPad Software)
  3. CellProfiler cell image analysis software (www.cellprofiler.org)

Procedure

  1. Leishmania culture, to generate amastigotes
    1. Thaw a vial with promastigotes and cultivate in 10 ml of 199 medium (see Recipes) supplemented with 10% of fetal bovine serum and 3 µg/ml of tunicamycin (selection marker for DsRed expression). Start a 1 x 106 parasites/ml culture in 80 cm2 T-flasks for 3 days at 26 °C. Parasites actively proliferate present > 90% viability on the third day.
    2. Centrifuge the culture 700 x g for 10 min in a 15 ml conical tube.
    3. Remove the supernatant and resuspend the parasites in 199 media (see Recipe 2) at a final concentration of 107 parasites/ml. Transfer the parasites to a T-flask.
    4. Incubate the culture at 34 °C for 3-4 days to allow promastigote to amastigote differentiation. Parasites with amastigote morphology (oval shape and no extracellular flagellum) are seen as early as 1 day after differentiation induction. However, the parasites require 3 days to complete differentiation.
      Note: Axenic amastigotes are commonly used in research, but they are not identical to intracellular amastigotes (Gupta et al., 2001).
    5. Centrifuge at 700 x g for 10 min. Wash with 10 ml PBS. Repeat this wash once.
    6. Transfer 1 x 108 parasites to a culture of Balb/c BMDM cultivated in 175 cm2 T-flasks (2 x 107 BMDM/flask).
    7. Keep the cultures at 34 °C and feed the cells with fresh RPMI 1640 supplemented with 10% FBS every 5 days.

  2. Harvesting amastigotes from BMDMs
    1. Remove the media from infected culture, add 5 ml of PBS and detach the cells using a cell scraper after one week of infection. Transfer the cell suspension to an ice cold glass Dounce tissue grinder and move the rod up and down 20 times to disrupt the cells, applying light pressure while maintaining the glass Dounce on ice. A large number of amastigotes are found in large parasitophorous vacuoles (Figure 1A), however the continuous parasite proliferation kills macrophages and gradually reduces parasite yield after purification.


      Figure 1. Intracellular L. amazonensis amastigotes isolation from macrophages. A. Balb/c BMDM infected with axenic amastigotes for 7 days, amastigotes expressing DsRed reside in large parasitophorous vacuoles. B. Clean amastigotes harvested from infected BMDMs. Scale bars = 10 μm.

    2. Transfer the lysate to a 15 ml conical tube, complete the volume to 13 ml with cold PBS.
    3. Centrifuge at 210 x g for 8 min, 4 °C and transfer the supernatant to a fresh 15 ml conical tube. Discard the pellet, which contains intact macrophages and large debris.
    4. Centrifuge the harvested supernatant at 675 x g for 12 min, 4 °C.
    5. Discard the supernatant and resuspend the pellet in 3 ml of 25% Percoll cushion (750 μl of Percoll, 300 μl of FBS and 1.95 ml of RPMI 1640).
    6. Centrifuge at 3,000 x g for 15 min, 4 °C.
    7. Remove the supernatant with a serological pipette, resuspend the pellet in 3 ml of cold PBS and centrifuge at 800 x g for 12 min to wash the parasites.
    8. Resuspend the parasites in 1 ml of RPMI and count the amastigotes using a hemocytometer. 5-10 x 107 parasites should be obtained in a typical preparation. Figure 1B shows amastigotes obtained using this protocol.
    9. Transfer the suspension of parasites to a micro-centrifuge tube and centrifuge at 800 x g for 12 min.
    10. Resuspend the pellet in PBS to a concentration of 1.25 x 109 parasites/ml for fly injections.

  3. Fly injections
    1. Use 7 to 10 days old flies grown in wide fly vials (25 flies/vial) at 25 °C.
    2. Keep animals at 29 °C for 3 days before infection to optimize the UAS-GAL4 system efficiency.
    3. Prepare the needles using a capillary puller and measure the diameter of the tip using an electronic caliper, break the capillary needle to the diameter of approximately 60 μm. One quick tip is to make needles with approximately the diameter of the femur of the fly first pair of legs.
    4. Assemble the capillary needle to the Nanoject (Drumond) following manufacturer’s instructions.
      Note: Use appropriate personal protective equipment and extreme caution to avoid accidents with the sharp glass needles.
    5. Vortex the parasite suspension obtained in Procedure B for few seconds and load approximately 2 mm of the needle.
    6. Anesthetize the flies in a CO2 pad under a stereomicroscope.
    7. Adjust the injector to dispose of 32.2 nl in each injection. Load the microinjector with parasite suspension and inject the parasites in the lateral side of the fly, between the first and second abdominal segments (Video 1). Monitor the injection checking the expansion of the abdomen. Discard the flies that did not have noticeable inflation of their abdomen after 5 sec of injection.
    8. Transfer the injected insects to a fresh vial and close it tightly.
    9. Change the needle before starting a new vial to avoid cross contamination between flies of different vials.
    10. Prepare control groups injected with PBS and uninjected for each fly strain to unbiasedly evaluate the effect of infection on survival.

      Video 1. Injection of flies. This video demonstrates the injection of 32 nl of a 1% bromophenol blue solution (for better visualization) in adult flies using a microinjector. The dye solution enters the hemocoel and expands the abdomen.

  4. Quantification method 1: Survival assay
    Wild-type flies are mildly susceptible to L. amazonensis infection, with 70-80% survival over the course of a 15-day experiment, but susceptible strains might present uncontrolled parasite proliferation in the hemolymph and reduced survival (Okuda et al., 2016).
    1. Ideally, use at least 50 flies per condition, for statistical power. Distribute the 50 flies in 2 vials with cornmeal fly food. The yellow color of this meal allows easy detection of dead flies.
    2. Monitor for dead flies once daily for 10-15 days post-infection. Move surviving flies to new vials every 2-3 days or when many dead flies are sitting on the bottom of the vial, obscuring the counting process.
    3. Plot the data in Prism GraphPad using the survival function. The Kaplan-Meier survival curves of experimental flies should be compared to the same flies injected with PBS and to WT flies injected with the same preparation of parasites. The comparison can be performed using Log-rank (Mantel-Cox) test. Figure 2 shows the result of a typical survival experiment where flies lacking phagocytes also known as Phagoless (HmlΔGal4-eGFP, UAS-Bax, Defaye et al., 2009) are more susceptible than either WT flies challenged with parasites or Phagoless flies injected with PBS.


      Figure 2. Survival of flies injected with L. amazonensis amastigotes. Flies devoid of hemocytes (Phagoless) present reduced resistance to Leishmania infection compared to wild-type flies. N = 60, statistical significance determined by Log-rank (Mantel-Cox test).

  5. Quantification method 2: Parasite burden determination by limiting dilution
    In this assay, infected flies are homogenized and a fraction of the suspension is serially diluted in Schneider’s insect medium, cultivated for 7 days and the presence of proliferating promastigotes evaluated microscopically.
    1. Fill three 96-well plates of with 100 μl of Schneider’s insect medium supplemented with 10% FBS, 50 U/ml of a penicillin/streptomycin solution, 10 µg/ml tetracycline and 5 µg/ml of tunicamycin (DsRed parasites are tunicamycin resistant). Add an additional 80 µl of media to the first row of one plate that will be the first dilution well. The high dose of antibiotics is essential to impair the growth of the bacteria present in the flies.
    2. Inside the hood, using a fine tweezer, pick one fly from the CO2 pad and quickly dip it into 70% ethanol, and then dip twice in sterile water. Put the clean fly in a 1.5 ml micro-centrifuge tube containing 200 µl of PBS.
    3. Gently grind the fly for 10 sec using a pellet grinder to disrupt the exoskeleton and release parasites from the hemolymph and tissues.
    4. Wait 5 min to settle the large tissues in the bottom and transfer 20 µl of the top fly homogenate into the first well in triplicate.
    5. Using a multichannel pipette make the serial dilution by transferring 100 µl of one well to the next well down the column, for 24 dilutions (until all the wells of the 3 plates are utilized). Change the pipette tips in each of the first 5 dilutions and every 3 dilutions afterwards to avoid carryover of parasites in the tips.
    6. After 7 days at 27 °C, check for the presence of live promastigotes under a light microscopy. Count the number of positive wells in each series and convert the value to the dilution factor (first well = 1/20 of one fly).
      Tip: During incubation, keep one side of the plate about 5 mm higher so the parasites will concentrate in the same side of the well to facilitate the identification of positive wells.

  6. Quantification method 3: Direct visualization of parasites from infected flies
    This method can be used to estimate the parasite burden of flies infected with parasites expressing fluorescent proteins such as DsRed.
    1. Gently grind a single fly in 40 µl of Schneider’s insect medium in a 1.5 ml micro-centrifuge tube using a pellet grinder for 10 sec and let it stand for 5 min to precipitate large debris. We suggest using at least 10 flies per sample for statistical power.
    2. Load 2 improved Neubauer or similar disposable counting chambers with the top suspension.
    3. Image the DsRed-expressing parasites using a fluorescence microscope with a 10x objective. Take 4 images per chamber for parasite counting (8 images in total).
    4. Using a cell image analysis software such as the open source CellProfiler (Carpenter et al., 2006) adjust the settings to precisely identify the parasites according to the size and fluorescence. An example of a simple pipeline for Cell Profiler is shown in Figure 3.
    5. Multiply the average number of parasites per image by 1.82 x 104 to calculate the number of parasites per ml, then multiply by 0.04 (40 μl total volume) to calculate the total number of parasites per fly. Adjust this formula if using a different imaging setup, considering the actual size of the image and the depth of the counting chamber.


      Figure 3. Counting parasites in fly homogenates using CellProfiler. The homogenate of a fly infected for 2 days was loaded into a counting chamber and imaged using a fluorescence microscope. The images were analyzed using Cell Profiler software. The left window shows the settings used to identify the parasites in the image. The right window shows the input raw image, the image with the identified parasites (colored) and, at the bottom, the image highlights all the identified objects (parasites are circled in green).

Data analysis

  1. For fly survival experiments, experimental flies are compared to wild-type flies with the same genetic background injected with same parasite preparation and to flies injected with PBS (vehicle). This detail is important because some mutant flies can be susceptible to injury and die after the injection of PBS. The statistical significance is determined by Log-rank (Mantel-Cox test).
  2. For parasite load determination by limiting dilution and by direct counting, the replicates of each 10 experimental flies are compared to WT flies infected with the same parasite preparation using One-way ANOVA.
  3. We perform at least 3 independent experiments for in vivo assays. Additionally, fly lines presenting phenotype of interest are validated by 3 additional independent assays.
  4. Statistical test was performed with GraphPad Prism7. Freeware such as R can also be used to perform this type of analysis.

Recipes

  1. 199 media to grow promastigotes
    199 media supplemented with:
    10% FBS
    20 mM HEPES
    0.35 g/L sodium bicarbonate
    5 μg/ml hemin (from a stock solution of 0.2% hemin in 0.1 N NaOH), pH 7.4
  2. 199 media to induce promastigote to axenic amastigote differentiation
    199 media supplemented with:
    20% FBS
    20 mM HEPES
    0.35 g/L sodium bicarbonate
    5 μg/ml hemin (from a stock solution of 0.2% hemin in 0.1 N NaOH)
    40 mM sodium succinate
    0.5% tryptic soy broth, pH 5.4

Acknowledgments

This protocol has been adapted from Okuda et al., 2016. Supported by the NIH (R21AI109678) to NS. The authors declare that there is no conflict of interest.

References

  1. Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2): 401-415.
  2. Carpenter, A. E., Jones, T. R., Lamprecht, M. R., Clarke, C., Kang, I. H., Friman, O., Guertin, D. A., Chang, J. H., Lindquist, R. A., Moffat, J., Golland, P. and Sabatini, D. M. (2006). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7(10): R100.
  3. Chen, R. (2013). Isolation and culture of mouse bone marrow-derived macrophages (BMM'phi'). Bio Protoc e68.
  4. Defaye, A., Evans, I., Crozatier, M., Wood, W., Lemaitre, B. and Leulier, F. (2009). Genetic ablation of Drosophila phagocytes reveals their contribution to both development and resistance to bacterial infection. J Innate Immun 1(4): 322-334.
  5. Dutta, S., Ray, D., Kolli, B. K. and Chang, K. P. (2005). Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 49(11): 4474-4484.
  6. Gupta, N., Goyal N. and Rastogi, A. K. (2001). In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 17(3) 150-153.
  7. Okuda, K., Tong, M., Dempsey, B., Moore K.J., Gazzinelli, R.T. Silverman, N. (2016). Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation. PLoS Pathog 12(6): e1005669.

简介

该协议描述了如何产生和收获无抗体的L型。 amazonensis amastigotes,以及如何用这些寄生虫感染成年果蝇(Drosophila melanogaster)。 这个模型概括了利什曼原虫和动物吞噬细胞之间相互作用的关键方面。

【背景】由利什曼原虫引起的利什曼病,根据宿主的寄生虫种类和免疫状态,影响皮肤,粘膜或内脏器官。 尽管小鼠感染模型已经提供了我们对这种寄生虫感染的大部分了解,但它并不适合于大规模的探索性方法。 在我们的实验室,我们利用果蝇黑腹果蝇的易处理和强大的遗传学来建立利什曼原虫感染的模型并进行小规模的筛选来鉴定涉及的宿主基因 在这些寄生虫的吞噬中(Okuda等人,2016)。 具体而言,使用UAS-GAL4表达系统(Brand和Perrimon,1993),可能与吞噬作用相关的一组基因被特异性敲低,监测突变体感染的苍蝇的存活和寄生物负荷以鉴定 调节感染的因素。

关键字:黑腹果蝇, 利什曼虫, 吞噬作用, 浆细胞, 血细胞, 纯培养无鞭毛体, 无鞭毛体, RNAi 筛选

材料和试剂

  1. 15ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339650)。
  2. (Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:178905)。细胞培养瓶(T-烧瓶)80cm2
  3. (Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:178883)。
  4. 细胞刮刀(Thermo Fisher Scientific,Thermo Scientific TM,目录号:179707)
  5. 10ml血清移液管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:170356)
  6. 1.5ml微量离心管(Corning,Costar ,产品目录号:3622)
  7. 96孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:167008)
  8. 移液器吸头(Fisher Scientific,目录号:21-402-269,21-402-569,21-402-589)
    制造商:Thermo Fisher Scientific,目录号:3752RI,3782RI。
  9. 一次性计数室(Bio-Rad Laboratories,目录号:1450003)
  10. 颗粒杵(Fisher Scientific,目录号:12-141-364)
  11. 飞蝇瓶(Genesee Scientific,Flystuff,目录号:32-116SB)
  12. (Leishmania amazonensis)(菌株RAT / BA / LV78,Dutta等人,2005)。
  13. BALB / c骨髓来源的巨噬细胞(BMDM)
    注:材料和试剂在Chen,2013的Bio-protocol paper中描述。Balb / c小鼠从The Jackson Laboratories获得。一只小鼠的股骨和胫骨产生大约3×10 8巨噬细胞。
  14. 磷酸盐缓冲盐水(PBS)(Corning,目录号:21-031-CV)
  15. RPMI 1640(Corning,目录号:10-040-CV)
  16. Percoll(GE Healthcare,目录号:17-0891-01)
  17. 溴酚蓝(Fisher Scientific,目录号:BP115-25)
  18. 飞行食品(FlyStuff Nutri-Fly糖蜜配方或Nutri-Fly TM布卢明顿配方,Genesee Scientific,Flystuff,目录编号:66-116)
  19. 黄色玉米面飞蝇食品(Genesee Scientific,Flystuff,目录号:62-101)
  20. Schneider昆虫培养基(Thermo Fisher Scientific,Gibco TM,目录号:21720024)
  21. 青霉素/链霉素(Corning,目录号:30-002-Cl)
  22. Tetracyclin(Sigma-Aldrich,目录号:T7660)
  23. 衣霉素(Cayman Chemical,目录号:11445)
  24. 70%乙醇
  25. 199媒体(康宁,目录编号:90-050-PB)
  26. 热灭活的胎牛血清(FBS)(亚特兰大生物公司,目录号:S12450)
  27. HEPES(Sigma-Aldrich,目录号:H3784-100G)
  28. 琥珀酸钠(Sigma-Aldrich,目录号:S9637)
  29. Hemin(Sigma-Aldrich,目录号:H9039)
  30. 胰蛋白酶大豆肉汤(BD,目录号:211825)
  31. 199媒体生长promastigotes(见食谱)
  32. 199 promastigote媒体为axenic amastigote分化(见食谱)

设备

  1. 装备有用于15ml锥形管的摆动桶的离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM Legend TM RT) br />
  2. 培养箱(VWR 水夹套CO 2培养箱)
  3. 玻璃反弹与研磨室和聚四氟乙烯棒,清除0.004“至0.006”(DWK生命科学,惠顿,目录号码:358011)
  4. 血细胞计数器Hausser Scientific Bright-Line TM计数室(Hausser Scientific,目录号:3110)
  5. 毛细管拉拔器(NARISHIGE,型号:PC-100)
  6. 纳升注射器(Drummond Scientific,型号:Nanoject II)
  7. 用于Nanojet II的Drummond Scientific毛细血管(Pipette.com,目录号:3-000-203-G / X)
    制造商:Drummond Scientific,目录号:3-000-203-G / X。
  8. 电子卡尺(Mitutoyo,目录号:500-197-30)
  9. (Genesee Scientific,FlyStuff,目录号:59-114)
  10. 立体显微镜(尼康仪器,型号:SMZ645)
  11. 多道移液管(Thermo Fisher Scientific,Thermo Scientific TM,型号:Finnpipette TM F2)
  12. Pellet研磨机(Fisher Scientific,目录号:12-141-361)
  13. 精密镊子(Fisher Scientific,目录号:12-000-122)
  14. 配备成像系统(相机:Nikon Instruments,型号:Ds-Qi1,成像软件:NIS-Elements)的荧光显微镜(Nikon Instruments,型号:Eclipse E400)

软件

  1. 成像软件:NIS元素基础研究,显微镜成像软件(尼康仪器)
  2. GraphPad Prism7(GraphPad软件)
  3. CellProfiler细胞图像分析软件( www.cellprofiler.org

程序

  1. 利马尼亚文化,以产生无ama癖
    1. 解冻与鞭毛细胞的小瓶,并在补充10%胎牛血清和3微克/毫升衣霉素(DsRed表达的选择标记)10毫升199培养基(见食谱)培养。在26℃下,在80cm 2的T-烧瓶中开始1×10 6个寄生虫/ ml培养物3天。寄生虫积极繁殖现在>第三天90%的生存能力。

    2. 在15毫升锥形管中离心培养700毫克/克10分钟
    3. 去除上清液,并在199培养基中重新悬浮寄生虫(参见配方2),最终浓度为10 7寄生虫/ ml。将寄生虫转移到T型瓶。
    4. 将培养物在34℃培养3-4天,以使前鞭毛体分化为无鞭毛体。早在分化诱导后1天即可见到具有无鞭毛体形态的寄生物(卵形和无细胞外鞭毛)。但是,寄生虫需要3天才能完成分化。
      注意:Axenic amastigotes通常用于研究,但它们与细胞内的无鞭毛体不相同(Gupta et al。,2001)。
    5. 700×g离心10分钟。用10毫升PBS洗涤。重复洗一次。
    6. 将1×10 8个寄生虫转移到在175cm 2 T-烧瓶中培养的Balb / c BMDM培养物(2×10 7 BMDM /瓶)。
    7. 将培养物保持在34℃,并每5天用添加有10%FBS的新鲜RPMI 1640进料细胞。

  2. 从BMDMs收获amastigotes
    1. 从感染的培养液中取出培养基,加入5 ml的PBS,并在感染一周后用刮刀分离细胞。将细胞悬液转移到冰冷的玻璃Dounce组织研磨机上,上下移动棒20次以破坏细胞,施加轻微的压力,同时将玻璃Dounce保持在冰上。在大的寄生泡中发现大量的无鞭毛体(图1A),然而连续的寄生虫增殖杀死巨噬细胞并在纯化后逐渐降低寄生虫产量。


      图1.细胞内亚马逊L. amazonensis 从巨噬细胞中分离无A. A. balb / c BMDM感染无菌无鞭毛体7天,表达DsRed的无鞭毛体居于大的寄生液泡中。 B.清洁从感染的BMDMs收获的无辜者。比例尺= 10微米。

    2. 将裂解物转移至15ml锥形管中,用冷PBS将体积加至13ml。
    3. 在210℃下离心8分钟,4℃并将上清液转移到新鲜的15ml锥形管中。丢弃含有完整巨噬细胞和大碎片的颗粒。
    4. 将收获的上清液在675×g下离心12分钟,4℃。
    5. 弃去上清液,重悬于3ml 25%Percoll垫(750μlPercoll,300μlFBS和1.95ml RPMI 1640)中的沉淀。

    6. 3000×g离心15分钟,4℃。
    7. 用血清移液管移出上清液,将沉淀重悬于3ml冷PBS中,并在800xg离心12分钟以清洗寄生虫。
    8. 重悬在1毫升RPMI的寄生虫和计数使用hemocytometer的amastigotes。在典型的制备中应该获得5-10×10 7个寄生虫。图1B显示了使用这个协议获得的amastigotes。
    9. 将寄生虫悬浮液转移到微量离心管中,并在800×g下离心12分钟。
    10. 在PBS中重悬沉淀至浓度为1.25×10 9寄生虫/ ml,用于飞注射。

  3. 飞注射

    1. 使用7到10天的苍蝇生长在宽飞蝇瓶(25苍蝇/小瓶)在25°C。

    2. 在感染前将动物保持在29°C 3天,以优化UAS-GAL4系统效率
    3. 使用毛细管拉拔器准备针头并使用电子卡尺测量针头的直径,将毛细针头折断至直径约60μm。一个快速提示是用大约第一对腿的股骨直径做针。
    4. 按照制造商的说明将毛细血管针装配到Nanoject(Drumond)上。
      注意:使用适当的个人防护装备,并特别小心,以避免尖锐的玻璃针头发生意外。
    5. 将程序B中获得的寄生虫悬浮液涡旋几秒钟,并加载约2mm的针头。

    6. 在立体显微镜下的CO 2垫中麻醉苍蝇。
    7. 调整喷油器,每次喷射处理32.2 nl。将寄生虫悬浮液加载到显微注射器上,并在第一和第二腹部之间的苍蝇侧面注射寄生虫(视频1)。监测注射检查腹部的扩张。
      注射5秒钟后,丢弃腹部明显膨胀的苍蝇
    8. 将注射的昆虫转移到新鲜的小瓶中并密封。

    9. 在启动新的样品瓶之前更换针头,以避免不同样品瓶的苍蝇之间发生交叉污染
    10. 准备注射了PBS的对照组,并且每只苍蝇应注射未注射以公正地评价感染对存活的影响。

      视频1

  4. 定量方法1:生存测定
    野生型苍蝇轻微易受L型影响。亚马逊(amazonensis)感染,在15天的实验过程中具有70-80%的存活率,但是易感菌株可能在血淋巴中呈现不受控制的寄生虫增殖并且存活减少(Okuda等人, 2016)。
    1. 理想情况下,每个条件至少使用50个苍蝇,用于统计功效。在2个小瓶中分配50个苍蝇用玉米面飞食物。这餐的黄色可以很容易地检测到苍蝇。
    2. 在感染后10-15天监测死苍蝇每天一次。每隔2-3天将幸存的苍蝇移到新的小瓶中,或者当许多苍蝇坐在小瓶底部时,使计数过程变得模糊。
    3. 使用生存函数在Prism GraphPad中绘制数据。实验苍蝇的Kaplan-Meier生存曲线应该与用PBS注射的相同果蝇和用相同制备的寄生虫注射的WT苍蝇进行比较。比较可以使用Log-rank(Mantel-Cox)测试来执行。图2显示了典型的生存实验的结果,其中缺乏吞噬细胞的苍蝇也被称为Phago sup-less(HmlΔGal4-eGFP,UAS-Bax,Defaye等人,2009)更易受到用寄生虫或用PBS注射的Phago 苍蝇攻击的WT苍蝇的影响。


      图2.注射 L. amazonensis amastigotes的苍蝇的生存。与野生型苍蝇相比,没有血细胞的苍蝇(Phago
  5. 定量方法2:通过有限稀释确定寄生虫负荷
    在该测定中,将感染的苍蝇匀浆,并将一部分悬浮液连续稀释于Schneider's昆虫培养基中,培养7天,并在显微镜下评估增殖的前鞭毛体的存在。
    1. 用100μl补充有10%FBS,50U / ml青霉素/链霉素溶液,10μg/ ml四环素和5μg/ ml衣霉素(DsRed寄生物是衣霉素抗性的)的Schneider's昆虫培养基填充三个96孔板, 。添加一个额外的80微升媒体的第一行将是第一个稀释孔。
      高剂量的抗生素对于减少苍蝇中存在的细菌的生长至关重要
    2. 在发动机罩内,使用精密的镊子,从CO 2垫上挑一只苍蝇,迅速将其浸入70%的乙醇中,然后在无菌水中浸两次。将干净的苍蝇放入含有200μlPBS的1.5ml微量离心管中。

    3. 使用颗粒研磨机轻轻研磨苍蝇10秒,以破坏外骨骼并释放血淋巴和组织中的寄生虫。
    4. 等待5分钟以沉淀底部的大组织,并将20μl顶部苍蝇匀浆转移到第一个孔中一式三份。
    5. 使用多道移液器,通过将100μl的一个孔转移到下一个孔中进行24次稀释(直到使用3个板的所有孔),进行系列稀释。改变移液器吸头的前5个稀释度,然后每3个稀释度,以避免寄生虫在尖端遗留。
    6. 在27℃7天后,在光学显微镜下检查活的前鞭毛体的存在。计算每个系列中的正孔数量并将其转换为稀释因子(第一个孔=一次的1/20)。
      提示:培养过程中,保持培养板的一侧高出约5mm,这样寄生虫将集中在培养孔的同一侧,便于识别阳性孔。

  6. 定量方法3:从感染的苍蝇直接观察寄生虫
    这种方法可以用来估计苍蝇感染寄生虫表达荧光蛋白,如DsRed的寄生虫负担。
    1. 轻轻研磨一个苍蝇在40微升施耐德的昆虫介质在一个1.5毫升的微型离心管使用颗粒研磨机10秒,并让其静置5分钟沉淀大型碎片。我们建议每个样本使用至少10个苍蝇用于统计功效。
    2. 加载2改进Neubauer或类似的一次性计数商会与顶部悬挂。
    3. 使用具有10倍物镜的荧光显微镜对表达DsRed的寄生虫进行成像。每个房间4个图像进行寄生虫计数(共8张图像)。
    4. 使用诸如开源CellProfiler(Carpenter等人,2006)的细胞图像分析软件调整设置以根据大小和荧光精确地鉴定寄生虫。图3显示了一个简单的Cell Profiler管道的例子。
    5. 将每个图像的平均寄生虫数乘以1.82×10-4,计算每毫升寄生虫数,然后乘以0.04(总体积40μl),计算每只寄生虫的总数。如果使用不同的成像设置,请考虑图像的实际尺寸和计数室的深度来调整此公式。


      图3.使用CellProfiler计数飞行匀浆中的寄生虫。将感染2天的苍蝇匀浆加载到计数室中并使用荧光显微镜成像。使用Cell Profiler软件分析图像。左侧窗口显示用于识别图像中寄生虫的设置。右边的窗口显示输入的原始图像,带有已识别的寄生虫(彩色)的图像,并在底部,图像突出显示所有已识别的对象(寄生虫以绿色圈出)。

数据分析

  1. 对于苍蝇存活实验,将实验苍蝇与具有相同遗传背景的相同寄生物制剂注射的野生型果蝇进行比较,并将苍蝇注射PBS(载体)。这个细节是重要的,因为一些突变苍蝇可能容易受伤,并在注射PBS后死亡。统计显着性由Log-rank(Mantel-Cox测试)确定。
  2. 对于通过有限稀释和直接计数确定的寄生虫负荷,使用单向ANOVA将每10个实验苍蝇的重复与感染相同寄生虫制剂的WT苍蝇进行比较。
  3. 我们进行至少3次独立实验用于体内实验。此外,表现出感兴趣表型的苍蝇线通过3个另外的独立分析进行验证。
  4. 用GraphPad Prism7进行统计学检验。 R等免费软件也可以用来执行这种类型的分析。

食谱

  1. 199媒体增长promastigotes
    199媒体补充:
    10%FBS
    20毫米HEPES
    0.35克/升碳酸氢钠
    5μg/ ml氯化血红素(来自0.1N NaOH中的0.2%血红素的储备溶液),pH7.4
  2. 199媒介诱导promastigote到axenseic amastigote分化
    199媒体补充:
    20%FBS
    20毫米HEPES
    0.35克/升碳酸氢钠
    5μg/ ml氯化血红素(来自在0.1N NaOH中的0.2%血红素的储备溶液)
    40毫克琥珀酸钠
    0.5%胰蛋白大豆肉汤,pH 5.4

致谢

该协议已被改编自Okuda等人,2016年。由NIH(R21AI109678)支持NS。作者声明不存在利益冲突。

参考

  1. Brand,A.H。和Perrimon,N。(1993)。 靶向基因表达作为改变细胞命运和产生显性表型的手段。 开发 118(2):401-415。
  2. 木匠,AE,琼斯,TR,Lamprecht,MR,克拉克,康,IH,Friman,O.,Guertin,DA,Chang,JH,Lindquist,RA,Moffat,J.,Golland,P.和Sabatini, DM(2006)。 CellProfiler:用于鉴定和定量细胞表型的图像分析软件 Genome Biol 7(10):R100。
  3. 陈,R.(2013)。 小鼠骨髓巨噬细胞(BMM'phi')的分离和培养 Bio Protoc e68。
  4. Defaye,A.,Evans,I.,Crozatier,M.,Wood,W.,Lemaitre,B。和Leulier,F。(2009)。 果蝇吞噬细胞的遗传消融揭示了它们对发育和抗性的贡献细菌感染。 J Innate Immun 1(4):322-334。
  5. Dutta,S.,Ray,D.,Kolli,B.K。和Chang,K.P.(2005)。 利用铝酞菁在细胞外和细胞内两个阶段对亚马逊利什曼原虫进行光动力学致敏氯化物在体外光解。 Antimicrob Agents Chemother 49(11):4474-4484。
  6. Gupta,N.,Goyal N.和Rastogi,A.K。(2001)。 利什曼原虫无菌无鞭毛体的体外培养和表征< / em>。趋势Parasitol 17(3)150-153。
  7. Okuda,K.,Tong,M.,Dempsey,B.,Moore K.J.,Gazzinelli,R.T. Silverman,N。(2016)。 利什曼原虫amazonensis 使CD36驱动寄生泡的成熟。 PLoS Pathog 12(6):e1005669。
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引用:Okuda, K. and Silverman, N. (2017). Drosophila Model of Leishmania amazonensis Infection. Bio-protocol 7(23): e2640. DOI: 10.21769/BioProtoc.2640.
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