Isolation and Infection of Drosophila Primary Hemocytes
果蝇原代血细胞的分离和感染
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2300 浏览次数: 10088
评审: Ruth A. FranklinBenoit ChassaingRamalingam Bethunaickan
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参见作者原研究论文
The authors used this protocol in:
Aug 2016
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
- Falcon tubes (15 ml) (Corning, Falcon®, catalog number: 352196 )
- Eppendorf tubes
- Cover glass, No 1.5, 22 mm2 (Corning, catalog number: 2850-22 )
- Petri dish (100 x 15 mm) (Corning, catalog number: 351029 )
- Kimwipe
- Micro slides (Corning, catalog number: 2948-75X25 )
- Sterile filter unit: 0.22 µm cellulose acetate filter flasks (Corning, catalog number: 430769 )
- Drosophila melanogaster wandering third instar larvae (Ashburner, 1989)
- E. coli (DH5α) constitutively expressing GFP
- peGFP (https://www.addgene.org/vector-database/2485/)
- pET-GFP-C11 (http://www.addgene.org/30183/)
- peGFP (https://www.addgene.org/vector-database/2485/)
- Bucket of ice
- LB growth media (Fisher Scientific, catalog number: BP1425-500 )
- Schneider’s Drosophila medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720 ) with 10% FBS (Atlanta Biologicals, Advantage, catalog number: S11095 )
- S2 cell media
- PBSS = 0.3% Saponin (Sigma-Aldrich, catalog number: S7900 ) in PBS
- 10% NGS
- Phalloidin Alexa 594 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A12381 )
- Vectashield (Vector Laboratories, catalog number: H-1000 )
- Clear nail polish
- Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
- Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
- Hydrochloric acid (HCl)
- Sodium hydroxide (NaOH)
- Paraformaldehyde (Electron Microscopy Sciences, catalog number: 19208 )
- 10% normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 ) in PBSS
- 10x PBS (see Recipes)
- 8% paraformaldehyde (see Recipes)
- Fixative: 4% paraformaldehyde in PBS (see Recipes)
Equipment
- Spectrophotometer (Molecular Devices, model: SpectraMax M2 )
- Low speed centrifuge for 15 ml Falcon tubes (Eppendorf, model: 5804 R )
- Dissecting stereomicroscope with Leica L2 cold light source (Leica Microsystems, model: Leica L2 )
- Fine dissecting forceps (Fine Scientific Tools)
- 37 °C incubator with shaking (Eppendorf, New BrunswickTM, model: Innova® 44 )
- 25 °C incubator (BioCold Environment, model: BC49-IN )
- Dissecting dish
- Confocal Microscope
Note: We use a Zeiss LSM510 (Zeiss, model: LSM510 ) with 63 x NA1.4 objective.
Software
- ImageJ (NIH)
- Prism (GraphPad)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
免疫学 > 动物模型 > 果蝇
微生物学 > 微生物-宿主相互作用 > 细菌
细胞生物学 > 细胞分离和培养 > 细胞分离
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