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Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells
卡他莫拉菌在呼吸道上皮细胞的粘附试验   

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Abstract

Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract mucosal epithelium. This protocol describes a method to measure adherence of M. catarrhalis to epithelial cell lines in vitro.

Keywords: Moraxella catarrhalis(卡他莫拉菌), Epithelial cells(上皮细胞), Adhesion(粘附), Respiratory tract(呼吸道), Pathogenesis(发病机制)

Materials and Reagents

  1. Human pharyngeal epithelial cell line Detroit 562 (ATCC, catalog number: CCL-138 )
  2. Type II alveolar epithelial cell line A549 (ATCC, catalog number: CCL-185 )
  3. DMEM + GlutaMAXTM-I (Life Technologies, Invitrogen™, catalog number: 31966-047 )
  4. Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758093S5403 )
  5. Trypsin-EDTA (0.25%-1 mM) (Life Technologies, catalog number: 25300-054 )
  6. Brain heart infusion (BHI) (Becton Dickinson and Company, catalog number: 237500 ) broth and BHI agar plates
  7. Antibiotics: spectinomycin or kanamycin (Merck KgaA, Calbiochem, catalog numbers: 567570-10 and 420311-5 )
  8. Bovine skin gelatin (Sigma-Aldrich, catalog number: G9382-100G )
  9. Glycerol (Merck KgaA, catalog number: 1.04092.1000)
  10. PBS without Ca2+ and Mg2+ (Westburg BV, catalog number: BE17-516F/12 )
  11. Saponin (Sigma-Aldrich, catalog number: 47036-50G-F )

Equipment

  1. 24-well tissue culture plate (Falcon®, catalog number: 353407 )
  2. 75 cm2 culture flask
  3. Centrifuge
  4. CO2 incubator
  5. Benchtop Incubator Shaker

Procedure

  1. The human pharyngeal epithelial cell line Detroit 562 and the type II alveolar epithelial cell line A549 were routinely grown in DMEM with GlutaMAXTM-I and 10% FCS at 37 °C and 5% CO2.
  2. For passage or seeding purposes epithelial cells were washed once in 10 ml PBS and detached using trypsin-EDTA as follows: add 3 ml Trypsin-EDTA per 75 cm2 culture flask, incubate 5 min at 37 °C and 5% CO2 until cells have detached from the bottom, collect cells with 7 ml culture medium, count cells, spin required amount for 5 min at 400 x g, resuspend pellet in required volume of culture medium.
  3. Two days prior to the adherence assay, 2 x 105 Detroit 562 cells per well were seeded into a 24-well tissue culture plate, and after one day, the growth medium was refreshed.
  4. A549 cells were seeded into 24-well tissue culture plates one day prior to the assay at 4 x 105 cells.
  5. For both cell lines, monolayers of approximately 1 x 106 cells per well were used for adherence assays.
  6. M. catarrhalis strains were inoculated on brain heart infusion (BHI) plates (supplemented with antibiotics when required) and grown overnight at 37 °C in an atmosphere containing 5% CO2.
  7. Bacteria were harvested from plate and resuspended in PBS supplemented with 0.15% gelatin (PBS-G).
  8. This suspension was used to inoculate BHI broth to an OD620 nm of ~ 0.05 and grown at 37 °C at 200-250 rpm until OD620 nm of 1.0 to 1.2. Subsequently, glycerol was added to a final concentration of 20%, and 1-ml aliquots were stored at -80 °C.
  9. Before each assay, bacteria were thawed on ice, washed once in 1 ml DMEM with GlutaMAXTM-I and 1% FCS (infection medium) and resuspended in the infection medium to 1 x 107 CFU ml-1.
  10. Epithelial cells were washed twice with 1 ml PBS, infected with 1 ml of the M. catarrhalis suspension (multiplicity of infection, 10 bacteria per cell), centrifuged for 5 minutes at 200 x g to facilitate contact between bacteria and cells, and incubated 1 h at 37 °C in a 5% CO2 environment.
  11. Non-adherent bacteria were removed by 3 washes with 1-ml PBS (PBS was added and subsequently carefully aspirated, no agitation). Detroit 562 or A549 cells were detached and lysed by addition of 1 ml 1% saponin in PBS-G followed by incubation at 37 °C and 5% CO2  for 10 min.
  12. CFUs were enumerated by plating 10-fold serial dilutions on BHI plates supplemented with the appropriate antibiotics. The percentage adherence of the mutants was calculated as the fraction of the inoculum that bound to the Detroit 562 cells.

Acknowledgments

This protocol was published in: de Vries et al. (2013). This study was financially supported by Vienna Spot of Excellence (VSOE) grant (ID337956).

References

  1. de Vries, S. P., Burghout, P., Langereis, J. D., Zomer, A., Hermans, P. W. and Bootsma, H. J. (2013). Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions. Mol Microbiol 87(1): 14-29.   

简介

卡他莫拉菌(Moraxella catarrhalis)是一种人类限制性病原体,其负责呼吸道感染,例如成年儿童中耳炎(OM)和慢性阻塞性肺病(COPD)的加重。 成功殖民和感染由M. 卡他性取决于其附着到呼吸道粘膜上皮的能力。 该协议描述了测量M的依从性的方法。 卡他莫利斯到上皮细胞系。

关键字:卡他莫拉菌, 上皮细胞, 粘附, 呼吸道, 发病机制

材料和试剂

  1. 人咽癌上皮细胞系Detroit 562(ATCC,目录号:CCL-138)
  2. II型肺泡上皮细胞系A549(ATCC,目录号:CCL-185)
  3. DMEM + GlutaMAX TM -I(Life Technologies,Invitrogen TM,目录号:31966-047)
  4. 胎牛血清(FCS)(Greiner Bio-One GmbH,目录号:758093S5403)
  5. 胰蛋白酶-EDTA(0.25%-1mM)(Life Technologies,目录号:25300-054)
  6. 脑心浸液(BHI)(Becton Dickinson and Company,目录号:237500)肉汤和BHI琼脂平板
  7. 抗生素:壮观霉素或卡那霉素(Merck KgaA,Calbiochem,目录号:567570-10和420311-5)
  8. 牛皮明胶(Sigma-Aldrich,目录号:G9382-100G)
  9. 甘油(Merck KgaA,目录号:1.04092.1000)
  10. PBS(不含Ca 2+和Mg 2+)(Westburg BV,目录号:BE17-516F/12)。
  11. 皂苷(Sigma-Aldrich,目录号:47036-50G-F)

设备

  1. 24孔组织培养板(Falcon ,目录号:353407)
  2. 75cm 2培养瓶
  3. 离心机
  4. CO <2>孵化器
  5. 台式孵化器

程序

  1. 人类咽上皮细胞系Detroit 562和II型肺泡上皮细胞系A549在37℃和5%CO 2下常规地生长在具有GlutaMAX TM sup-1和10%FCS的DMEM中。
  2. 为了通过或接种目的,将上皮细胞在10ml PBS中洗涤一次,并如下使用胰蛋白酶-EDTA分离:每75cm 2培养瓶加入3ml胰蛋白酶-EDTA,在37℃孵育5分钟和5%CO 2,直到细胞从底部分离,用7ml培养基收集细胞,计数细胞,在400×g下旋转所需量5分钟,重悬沉淀在所需体积的培养基中。
  3. 在粘附测定前两天,将2×10 5个底特律562细胞/孔接种到24孔组织培养板中,一天后,更新生长培养基。
  4. 将A549细胞在4×10 5个细胞的测定前一天接种到24孔组织培养板中。
  5. 对于两种细胞系,将每孔大约1×10 6个细胞的单层用于粘附测定。
  6. M。卡那霉素菌株接种在脑心浸液(BHI)板(当需要时补充有抗生素)中,并在37℃下在含有5%CO 2的气氛中生长过夜。
  7. 从板中收获细菌并重悬于补充有0.15%明胶(PBS-G)的PBS中。
  8. 将该悬浮液用于接种BHI肉汤至〜550nm的OD 620nm,并在37℃下以200-250rpm生长,直到OD 620nm为1.0-1.2。随后,加入甘油至终浓度为20%,将1ml等分试样储存在-80℃。
  9. 在每次测定之前,将细菌在冰上解冻,在1ml含有GlutaMAX TM和1%FCS(感染培养基)的DMEM中洗涤一次,并在感染培养基中重悬浮至1×10 7 CFU ml -1
  10. 上皮细胞用1ml PBS洗涤两次,用1ml的M感染。卡那霉素悬浮液(感染复数,每个细胞10个细菌),在200×g离心5分钟以促进细菌和细胞之间的接触,并在37℃下在5℃下孵育1小时%CO 2环境。
  11. 通过用1ml PBS洗涤3次(加入PBS,随后小心地吸出,不搅动)除去非粘附细菌。 将底特律562或A549细胞分离并通过加入1ml 1%的皂甙在PBS-G中的溶液裂解,然后在37℃和5%CO 2中孵育。 10分钟。
  12. 通过在补充有合适的抗生素的BHI板上铺板10倍系列稀释物来计数CFU。 突变体的粘附百分比计算为结合到底特律562细胞的接种物的分数。

致谢

该协议发表于:de Vries et al。(2013)。 本研究得到了维也纳卓越奖(VSOE)资助(ID337956)的资助。

参考文献

  1. de Vries,S.P.,Burghout,P.,Langereis,J.D.,Zomer,A.,Hermans,P.W.和Bootsma,H.J。(2013)。 在铁限制条件下对粘膜炎莫拉氏菌生长的遗传学要求。 Mol Microbiol 87(1):14-29。   
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vries, S. P. and Bootsma, H. J. (2013). Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells. Bio-protocol 3(21): e956. DOI: 10.21769/BioProtoc.956.
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