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Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages
庆大霉素保护试验测定细菌在巨噬细胞中的存活能力   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Dec 2013

Abstract

Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.

Materials and Reagents

  1. RAW 264.7 cells (ATCC, TIB-71 )
  2. E. coli CFT073 or other strains of bacteria
  3. LB broth (Beckton Dickinson, catalog number: 244610 )
  4. Agar (Beckton Dickinson, catalog number: 214010 )
  5. RPMI1640 (Life Technologies, catalog number: 11875-093 )
  6. Gentamicin 50 mg/ml (Life Technologies, catalog number: 15750-102 )
  7. PBS (pH 7.4)
  8. LB plates (see Recipes)
  9. Certified, heat inactivated fetal bovine serum (FBS) (Life Technologies, catalog number: 10082139 ) (see Recipes)
  10. 100x penicillin, streptomycin and glutamine (PSG) (Life Technologies, catalog number: 10378-016 ) (see Recipes)
  11. Saponin (Sigma-Aldrich, catalog number: 8047-15-2) (see Recipes)

Equipment

  1. 24 well plates (Corning, Costar®, catalog number: 3524 )
  2. 0.22 micron filter (Millipore, catalog number: SCGP00525 )
  3. 37 °C, 5% CO2 incubator
  4. Sorvall Legend RT table top centrifuge
  5. Shimadzu UV-1601PC Spectrophotometer

Procedure

  1. Culture RAW 264.7 cells in RPMI1640 with 10% heat inactivated fetal bovine serum and 1x penicillin streptomycin and glutamine at 37 °C in a CO2 incubator.
  2. Count the cells using standard procedures and seed 2 x 105 RAW cells/well, by adding 1 ml per well in a 24 well plate. Set up 2 plates; one for T0 and another for T2 and incubate in a CO2 incubator.
  3. Start overnight cultures of CFT073 in 3 ml of LB broth in a 10 ml polypropylene tube and incubate at 37 °C shaking at 200 rpm. Use appropriate culture media when using other bacterial strains.
  4. Determine the multiplicity of infection (MOI = number of bacteria per RAW 264.7 cell) and alter the inocula preparation accordingly. For an MOI of 10, add 2 x 106 cfu of E. coli CFT073 for a well containing 2 x 105 RAW cells.
  5. Pellet the bacterial cells from the 3 ml culture by centrifuging at 4,000 rpm for 10 min in a Sorvall centrifuge. Resupend the pellet in 3 ml PBS.
  6. Adjust the OD600 of broth culture to 4 in PBS, by measuring the optical density of a 1:10 diluted culture in a spectrophotometer and calculating the optical density of undiluted culture.
  7. Dilute 1:100 in PBS.
  8. Dilute 1:10 in RPMI 1640 without antibiotics.
  9. Wash RAW cells twice with PBS.
  10. Add 1 ml of diluted culture to each well; three technical replicates per sample and three biological replicates of the entire experiment are recommended. Use the same set up for T0 and T2 plates.
  11. Incubate at 37 °C, 5% CO2 for 30 min.
  12. Aspirate supernatant and wash 3 times with PBS.
  13. Add 1 ml of RPMI 1640 with gentamicin (200 µg/ml) per well.
  14. Incubate the T0 plate at 37 °C, 5% CO2 for 15 min.
  15. Incubate the T2 plate at 37 °C, 5% CO2 for 2 h.
  16. After 15 min or 2 h, remove the supernatants entirely by aspiration.
  17. Lyse cells with 1 ml of filter sterilized 1% saponin in H2O per well by incubating for 10 min at room temperature followed by vigorous pipetting. Determine the extent of lysis using a standard inverted microscope.
  18. Plate undiluted and -2 dilution for T0 and T2 time points on LB plates.
  19. % Killing = [(T0-T2)/T0]*100

Recipes

  1. LB plates
    Add 15 g of agar to 1 L of LB broth
  2. FBS and PSG
    Added at 10% and 1x final concentration, respectively, to RPMI1640
  3. Saponin
    Dissolved in autoclaved double distilled water and filter sterilized using a 0.22 μM filter

Acknowledgments

This protocol was adapted from the publication: Subashchandrabose et al. (2013).

References

  1. Subashchandrabose, S., Smith, S. N., Spurbeck, R. R., Kole, M. M. and Mobley, H. L. (2013). Genome-wide detection of fitness genes in uropathogenic Escherichia coli during systemic infection. PLoS Pathog 9(12): e1003788.

简介

巨噬细胞是参与编制宿主防御感染的关键细胞。 在这里,我们描述的细菌杀伤测定在巨噬细胞,可以适应任何细菌病原体的协议。 使用该测定,我们分析了广泛使用的巨噬细胞系RAW 264.7细胞内的大肠杆菌(大肠杆菌)的野生型和突变株的存活。 可以使用该测定来描绘巨噬细胞内的细胞内存活缺陷的细菌突变体。

材料和试剂

  1. RAW 264.7细胞(ATCC,TIB-71)
  2. E。 大肠杆菌 CFT073或其他细菌菌株
  3. LB肉汤(Beckton Dickinson,目录号:244610)
  4. 琼脂(Beckton Dickinson,目录号:214010)
  5. RPMI1640(Life Technologies,目录号:11875-093)
  6. 庆大霉素50mg/ml(Life Technologies,目录号:15750-102)
  7. PBS(pH 7.4)
  8. LB板(见配方)
  9. 认证的热灭活胎牛血清(FBS)(Life Technologies,目录号:10082139)(参见配方)
  10. 100x青霉素,链霉素和谷氨酰胺(PSG)(Life Technologies,目录号:10378-016)(参见Recipes)
  11. 皂苷(Sigma-Aldrich,目录号:8047-15-2)(参见Recipes)

设备

  1. 24孔板(Corning,Costar ,目录号:3524)
  2. 0.22微米过滤器(Millipore,目录号:SCGP00525)
  3. 37℃,5%CO 2培养箱
  4. Sorvall Legend RT台式离心机
  5. Shimadzu UV-1601PC分光光度计

程序

  1. 将RAW264.7细胞在含有10%热灭活的胎牛血清和1×青霉素链霉素和谷氨酰胺的RPMI1640中在37℃下在CO 2培养箱中培养。
  2. 使用标准程序计数细胞,并通过在24孔板中每孔加入1ml将种子以2×10 5个RAW细胞/孔接种。 设置2块板; 一个用于T0,另一个用于T2,并在CO 2培养箱中孵育
  3. 开始在10毫升聚丙烯管中的3ml LB肉汤中的CFT073的过夜培养物,并在37℃下以200rpm振摇孵育。 使用其他细菌菌株时,使用适当的培养基
  4. 确定感染的多重性(MOI =每个RAW 264.7细胞的细菌数),并相应地改变接种制剂。 对于MOI为10,加入2×10 6 cfu的E。 大肠杆菌 CFT073用于含有2×10 5个 RAW细胞的孔。
  5. 通过在Sorvall离心机中以4,000rpm离心10分钟从3ml培养物中沉淀细菌细胞。 在3ml PBS中重新填充沉淀
  6. 通过在分光光度计中测量1:10稀释的培养物的光密度并计算未稀释的培养物的光密度,将肉汤培养物的OD 600调节至PBS中的4倍。
  7. 在PBS中稀释1:100
  8. 在无抗生素的RPMI 1640中稀释1:10
  9. 用PBS洗涤RAW细胞两次。
  10. 向每个孔中加入1ml稀释的培养物; 推荐每个样品三个技术重复和整个实验的三个生物学重复。 对T0和T2板使用相同的设置。
  11. 在37℃,5%CO 2孵育30分钟
  12. 吸出上清液,用PBS洗涤3次
  13. 每孔加入1ml含庆大霉素(200μg/ml)的RPMI 1640
  14. 将T0板在37℃,5%CO 2孵育15分钟
  15. 在37℃,5%CO 2孵育T2板2小时
  16. 15分钟或2小时后,通过抽吸完全去除上清液
  17. 通过在室温下孵育10分钟,随后强烈吸液,用1ml过滤灭菌的H 2 O 2 /孔中的1%皂苷溶解细胞。 使用标准倒置确定溶解的程度 显微镜
  18. 未稀释的板和在LB板上的T0和T2时间点的-2稀释
  19. %杀灭= [(T0-T2)/T0]×100

食谱

  1. LB平板
    将15g琼脂加入1L LB肉汤中
  2. FBS和PSG
    分别以10%和1x最终浓度加入RPMI1640
  3. 皂苷
    溶解在高压灭菌的双蒸水中,并使用0.22μM过滤器过滤灭菌

致谢

该协议改编自出版物:Subashchandrabose等人(2013)。

参考文献

  1. Subashchandrabose,S.,Smith,S.N.,Spurbeck,R.R.,Kole,M.M.and Mobley,H.L。(2013)。 全基因组检测uropathogenic中的适应基因 大肠杆菌在全身感染期间。

    9(12):e1003788。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Subashchandrabose, S. and Mobley, H. L. (2014). Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages. Bio-protocol 4(18): e1235. DOI: 10.21769/BioProtoc.1235.
  2. Subashchandrabose, S., Smith, S. N., Spurbeck, R. R., Kole, M. M. and Mobley, H. L. (2013). Genome-wide detection of fitness genes in uropathogenic Escherichia coli during systemic infection. PLoS Pathog 9(12): e1003788.
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