搜索

Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines
霍乱弧菌在真核细胞系粘附性的分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of different cell types. A protocol for adherence of V. cholerae to various cell lines is described. The adhered bacteria can be used to examine expression of genes that are differentially expressed between adhered and unadhered bacteria or other purposes (Dey et al., 2013).

Keywords: Adherence to INT407(坚持Int407), Vibrio cholerae adherence(霍乱弧菌粘附), Host pathogen interaction(宿主-病原体相互作用), Virulence gene induction(毒力基因的诱导), VieA induction(VieA感应)

Materials and Reagents

  1. Cell lines: INT 407, Hep-2, Hela, HT-29 (available from ATCC and other commercial sources)
  2. Vibrio cholerae classical biotype strain O395
  3. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®)
    1. High glucose (4,500 mg/L) (catalog number: 12800-017 )
    2. Low glucose (1,000 mg/L) (catalog number: 31600-034 )
    3. No glucose (catalog number: 11966-025 )
  4. New born calf serum (NCS) (not heat inactivated) (Life Technologies, Gibco®, catalog number: 16010-159 )
  5. 10x Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 15400 )
  6. TritonX-100 (Affymetrix, catalog number: T1001 )
  7. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: C6164 )
  8. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021 )
  9. D-Galactose
  10. 10x cell lysis buffer (Cell Signaling Technology, catalog number: 9803 )
  11. pNPP (5 mg) (Sigma Chemical, catalog number: N-9389 )
  12. Bacto tryptone (BD, DifcoTM, catalog number: 211705 )
  13. Bacto yeast extract (BD, DifcoTM, catalog number: 212750 )
  14. Bacto agar (BD, DifcoTM, catalog number: 214010 )
  15. NaHCO3 (Merck KGaA)
  16. NaCl (Merck KGaA)
  17. Na2HPO4 (Merck KGaA)
  18. KCl (Merck KGaA)
  19. KH2PO4 (Merck KGaA)
  20. 70% ethanol (Merck KGaA)
  21. NaOH (Merck KGaA)
  22. Personal protective items (apron, gloves, mask etc.)
  23. Bio-Rad protein assay dye reagent (Bio-Rad Laboratories)
  24. Complete DMEM (100 ml) (see Recipes)
  25. Incomplete DMEM (100 ml) (see Recipes)
  26. No glucose complete DMEM (100ml) (see Recipes)
  27. 10x PBS (500 ml, pH 7.4) (see Recipes)
  28. 1x PBS (50 ml) (see Recipes)
  29. 1x Trypsin-EDTA (10 ml) (see Recipes)
  30. 1% Triton X-100 (50 ml) (see Recipes)
  31. 0.9% saline solution (see Recipes)
  32. LB medium (100 ml) (see Recipes)
  33. LB Agar (100 ml) (see Recipes)
  34. Alkaline phosphatase assay buffer (10 ml) (see Recipes)

Equipment

  1. 25 cm2 tissue-culture treated flasks (BD Biosciences, Falcon®, catalog number: 353108 )
  2. Sterile, disposable 15 ml and 50 ml centrifuge tubes
  3. Sterile, disposable 1.5 ml microcentrifuge tubes
  4. Sterile microtips
  5. Sterile cryovials
  6. Cell scraper (BD Biosciences, Falcon®)
  7. 0.22 μm filter units (Millex-GP) (Millipore, catalog number: SLGP033RS )
  8. CO2 incubator
  9. Laminar flow hood
  10. Liquid nitrogen storage container
  11. Inverted microscope
  12. Centrifuge
  13. Microcentrifuge
  14. Pipetteman
  15. UV-Vis Spectrophotometer

Procedure

Note: For handling cell lines and bacterial cultures, aseptic techniques must be used and work should be carried out in a sterile laminar flow hood when practicable.

  1. Culturing of cell lines
    1. An aliquot of the chosen cell line (approx. 106 cells) stored in liquid nitrogen (see Note 1) was used to seed a 25 cm2 tissue culture flask containing 5 ml complete DMEM. The cultures were grown to 70-80% confluency (approx. 4 x 106 cells) at 37 °C in 5% CO2 humidified atmosphere. INT 407 was grown in low glucose complete DMEM, Hep-2, Hela and undifferentiated HT-29 were grown in high glucose complete DMEM. Differentiated (see Note 2) HT-29 was grown in no glucose complete DMEM at 10% CO2 humidified atmosphere.
    2. The cultures were washed three times with 5 ml 1x phosphate buffered saline (PBS) and 4 ml low glucose incomplete DMEM was added.

  2. Bacterial cell culture
    1. A loopful of V. cholerae strain O395 preserved at -70 °C in LB medium containing 20% glycerol was streaked on LB agar plates and incubated at 37 °C for 16-18 h.
    2. A loopful of V. cholerae culture from the plate was inoculated into 5 ml LB medium and grown with aeration (shaking at approx. 200 rpm in a rotary shaker) at 37 °C for 16-18 h. From this culture, a 1% inoculum (50 µl) was added to 5 ml LB medium and grown to the mid logarithmic phase (OD600 0.5) with shaking at 37 °C.
    3. The V. cholerae culture was centrifuged (5,000 x g, 5 min), washed with 5 ml 1x PBS, and suspended in low glucose incomplete DMEM at a concentration of 109 colony forming units (CFU) per ml (Bacterial cells from approximately 2 ml logarithmic phase cultures in LB medium were suspended in 1 ml incomplete DMEM.).

  3. Bacterial adherence to cell lines
    1. Cell lines grown to 70-80% confluency in 25 cm2 flasks were used.
    2. V. cholerae culture (200 µl, approximately 2 x 108 CFU) in low glucose incomplete DMEM was added to the 70-80% confluent cell lines, resultant multiplicity of infection (MOI) was about 50.
    3. After addition of bacteria, the cell lines were incubated for 45 min at 37 °C in 5% CO2 humidified atmosphere for optimum adherence.
    4. After 45 min, the supernatant was discarded and non-adhered bacteria were removed by washing the infected cell line 4 times with 5 ml 1x PBS and then low glucose complete DMEM (5 ml) was added and incubated for different periods of time (1-12 h).
    5. After the desired time period, the supernatant was discarded and the infected cell line was washed 4 to 5 times with 5 ml 1x PBS.
    6. 1 ml of 1% Triton X-100 was added and incubated for 2 min at room temperature with gentle swirling when the cell line could be seen to lyse and detach from the flask. The total culture was collected in 1.5 ml micro centrifuge tubes and vigorous pipetting or vortexing was done. The Triton X-100 treatment lysed the eukaryotic cells but had no effect on the viability of the bacterial cells (see Note 3).
    7. The bacterial cells were collected by centrifugation (5,000 x g, 5 min) at room temperature and the pellet was suspended in 1 ml 1x PBS.
    8. From this suspension 10 μl was added in 990 μl of 0.9% saline solution and bacterial CFU was determined by serial dilution and plating on LB agar plates. Remaining adhered bacterial cells could be used for gene expression studies or other purposes.
    9. Parallel experiments were performed with V. cholerae under identical conditions but without cell line as an unadhered control.

Notes

  1. Cell lines were stored in liquid nitrogen with 10% DMSO as a cryoprotectant.
  2. HT29 differentiation
    1. Differentiation of the HT29 cells was performed essentially as described by Huet et al. (1987).
      1. Undifferentiated HT29 cells were grown to 70-80% confluency in high glucose complete DMEM in 25 cm2 flasks.
      2. 1 ml of a solution containing 0.5% trypsin and 1 mM EDTA was added, after about 5 min the detached cells were used to seed a 25 cm2 flask containing no glucose complete DMEM supplemented with 20 mM glucose and 1 mM galactose (day 0).
      3. Then the following schedule was followed to gradually increase the concentration of galactose to 5 mM and decrease the concentration of glucose: (a) day 1, 15 mM glucose plus 1 mM galactose; (b) day 2, 10 mM glucose plus 3 mM galactose; (c) day 3, 5 mM glucose plus 4 mM galactose; (d) day 4, 5 mM galactose. The medium was changed every day over the period of 4 days. Once adapted, cells were grown for several passages in no glucose complete DMEM containing galactose (5 mM).
    2. Alkaline phosphatase (AP) activity was estimated as a marker of HT29 differentiation (Luongo et al., 2002).
      1. Differentiated and undifferentiated HT29 cells were released from the 70-80% confluent flask by treatment with 1 ml trypsin (0.5%) - EDTA (1 mM) solution, washed three times with 1 ml cold 1x PBS and lysed on ice with 1x cell lysis buffer.
      2. For AP activity assay, 50 μl cell lysate was incubated with 1 ml  p-nitrophenyl phosphate (1 mg/ml) in AP assay buffer for 30 min at room temperature and the reaction was stopped by adding 100 μl of 0.1 M K2HPO4.
      3. AP activity was calculated as absorbance at 405 nm (A405) per mg of total cellular protein. A405 was measured against lysates incubated without substrate.
      4. Total protein was estimated by Bradford method using the Bio-Rad protein assay dye reagent.
  3. To examine if 1% Triton X-100 treatment had any effect on bacterial cells, a bacterial culture (OD600 0.5 approximately) was divided into 2 equal parts, one part was treated with Triton X-100 for 2 min, and CFU of the treated and untreated culture was determined. If CFU of the two cultures are comparable, it may be assumed that 1% Triton X-100 treatment had no effect on the viability of the bacterial cells used in the study.

Recipes

  1. Complete DMEM (100 ml)
    1.0 g DMEM (low or high glucose)
    0.37 g sodium bicarbonate
    10 ml NCS (heat-inactivated for 30 min in a 56 °C water bath)
    Volume adjusted to 100 ml with deionised water
    Filtered using 0.22 μm filter units
    Stored at 4 °C
  2. Incomplete DMEM (100 ml)
    1.0 g DMEM (low glucose)
    0.37 g sodium bicarbonate
    Volume adjusted to 100 ml with deionised water
    Filtered using 0.22 μm filter units
    Stored at 4 °C
  3. No glucose complete DMEM (100 ml)
    89 ml liquid DMEM (no glucose)
    10 ml heat inactivated NCS
    Filtered using 0.22 μm filter units
    Stored at 4 °C
  4. 10x PBS (500 ml, pH 7.4)
    40.0 g NaCl
    1.0 g KCl
    6.07 g Na2HPO4
    1.0 g KH2PO4
    Volume adjusted to 500 ml with deionised water, if required pH adjusted with NaOH
    Autoclaved
  5. 1x PBS (50 ml)
    5 ml of 10x PBS diluted in 45 ml of deionised water
    Filtered using 0.22 μm filter units
  6. 1x Trypsin-EDTA (10 ml)
    1 ml of 10x Trypsin-EDTA diluted in 9 ml of sterile 1x PBS
    1 ml aliquots distributed in centrifuge tubes
    Stored at 4 °C
  7. 1% Triton X-100 (50 ml)
    0.5 ml 100% Triton X diluted in 49.5 ml of sterile 1x PBS
  8. 0.9% saline solution
    0.9 g NaCl in 100 ml deionised water
    Autoclaved
  9. LB medium (100 ml)
    1 g Bacto tryptone (1%)
    0.5 g Bacto yeast extract (0.5%)
    0.5 g NaCl (0.5%)
    Volume adjusted to 100 ml with deionised water, pH adjusted to 8.6 with NaOH
    Autoclaved
  10.  LB Agar (100 ml)
    100 ml LB medium
    1.5 g Bacto agar (1.5%)
    Volume adjusted to 100 ml with deionised water, pH adjusted to 7.4 with NaOH
    Autoclaved
  11. Alkaline phosphatase assay buffer (10 ml)
    1 ml 1 M Tris/HCl (pH 9.5) (final concentration 100 mM)
    50 µl 1 M MgCl2 (final concentration 5 mM)
    Volume adjusted to 10 ml with deionised water
    Autoclaved

Acknowledgments

This protocol is adapted from Huet et al. (1987); Luongo et al. (2002); and Dey et al. (2013).

References

  1. Dey, A. K., Bhagat, A. and Chowdhury, R. (2013). Host cell contact induces expression of virulence factors and VieA, a cyclic di-GMP phosphodiesterase, in Vibrio cholerae. J Bacteriol 195: 2004-2010.
  2. Huet, C., Sahuquillo-Merino, C., Coudrier, E. and Louvard, D. (1987). Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell Biol 105: 345-357.
  3. Luongo, D., Mazzarella, G., Della, R. F., Maurano, F. and Rossi, M. (2002). Down-regulation of ERK1 and ERK2 activity during differentiation of the intestinal cell line HT-29. Mol Cell Biochem 231: 43-50.

简介

霍乱弧菌(霍乱弧菌)殖民肠上皮并分泌霍乱毒素(CT),霍乱毒素(CT)是一种有效的肠毒素,其导致霍乱的特征性严重的流体损失。 V。 霍乱菌是一种非侵入性革兰氏阴性细菌,其粘附于肠细胞以及各种不同的细胞类型。 遵守 V的协议。 霍乱等对各种细胞系的影响。 粘附的细菌可用于检查在粘附和未粘附的细菌或其他目的之间差异表达的基因的表达(Dey等人,2013)。

关键字:坚持Int407, 霍乱弧菌粘附, 宿主-病原体相互作用, 毒力基因的诱导, VieA感应

材料和试剂

  1. 细胞系:INT 407,Hep-2,Hela,HT-29(可从ATCC和其它商业来源获得)
  2. 霍乱弧菌经典生物型菌株O395
  3. 将Dulbecco改良的Eagle's培养基(DMEM)(Life Technologies,Gibco )
    1. 高葡萄糖(4500mg/L)(目录号:12800-017)
    2. 低葡萄糖(1,000mg/L)(目录号:31600-034)
    3. 无葡萄糖(目录号:11966-025)
  4. 新生小牛血清(NCS)(未热灭活)(Life Technologies,Gibco ,目录号:16010-159)
  5. 10x胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:15400)
  6. TritonX-100(Affymetrix,目录号:T1001)
  7. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:C6164)
  8. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G7021)
  9. D-半乳糖
  10. 10×细胞裂解缓冲液(Cell Signaling Technology,目录号:9803)
  11. pNPP(5mg)(Sigma Chemical,目录号:N-9389)
  12. 细菌胰蛋白胨(BD,Difco TM ,目录号:211705)
  13. 细菌酵母提取物(BD,Difco ,目录号:212750)
  14. Bacto琼脂(BD,Difco TM,目录号:214010)
  15. NaHCO 3(Merck KGaA)
  16. NaCl(Merck KGaA)
  17. Na 2 HPO 4(Merck KGaA)
  18. KCl(Merck KGaA)
  19. (Merck KGaA)。
  20. 70%乙醇(Merck KGaA)
  21. NaOH(Merck KGaA)
  22. 个人防护用品(围裙,手套,面具等)
  23. Bio-Rad蛋白测定染料试剂(Bio-Rad Laboratories)
  24. 完成DMEM(100 ml)(参见配方)
  25. 不完全DMEM(100 ml)(参见配方)
  26. 无葡萄糖完全DMEM(100ml)(见配方)
  27. 10x PBS(500ml,pH7.4)(参见Recipes)
  28. 1x PBS(50ml)(见配方)
  29. 1x胰蛋白酶-EDTA(10ml)(参见配方)
  30. 1%Triton X-100(50ml)(参见配方)
  31. 0.9%盐水溶液(见配方)
  32. LB培养基(100ml)(参见配方)
  33. LB琼脂(100ml)(见配方)
  34. 碱性磷酸酶测定缓冲液(10ml)(参见配方)

设备

  1. 25cm 2组织培养处理的烧瓶(BD Biosciences,Falcon ,目录号:353108)。
  2. 无菌,一次性15 ml和50 ml离心管
  3. 无菌,一次性1.5 ml微量离心管
  4. 无菌微尖端
  5. 无菌cryovials
  6. 细胞刮刀(BD Biosciences,Falcon )
  7. 0.22μm过滤器单元(Millex-GP)(Millipore,目录号:SLGP033RS)
  8. CO <2>孵化器
  9. 层流罩
  10. 液氮储存容器
  11. 倒置显微镜
  12. 离心机
  13. 微量离心机
  14. Pipetteman
  15. 紫外可见分光光度计

程序

注意:为了处理细胞系和细菌培养物,必须使用无菌技术,并且如果可行,应在无菌层流罩中进行操作。

  1. 细胞系的培养
    1. 使用储存在液氮(参见注释1)中的所选择的细胞系(约10 6个细胞)的等分试样接种25cm 2组织培养瓶,其含有5 ml完全DMEM。在37℃,5%CO 2加湿气氛中,将培养物生长至70-80%汇合(约4×10 6个细胞)。 INT 407在低葡萄糖完全DMEM中生长,Hep-2,Hela和未分化HT-29在高葡萄糖完全DMEM中生长。分化(参见注释2)HT-29在10%CO 2加湿气氛下在无葡萄糖的完全DMEM中生长。
    2. 将培养物用5ml 1x磷酸盐缓冲盐水(PBS)洗涤三次,并加入4ml低葡萄糖不完全DMEM。

  2. 细菌细胞培养
    1. 一个循环的 V。在含有20%甘油的LB培养基中于-70℃保存的霍乱菌株O395在LB琼脂平板上划线并在37℃下孵育16-18小时。
    2. 一个循环的 V。霍乱弧菌培养物接种到5ml LB培养基中并在37℃下通气(在旋转振荡器中以约200rpm振摇)生长16-18小时。从该培养物中,将1%接种物(50μl)加入到5ml LB培养基中,并在37℃下振荡生长至中对数期(OD <600)。
    3. V。霍乱菌培养物离心(5,000×g,5分钟),用5ml 1x PBS洗涤,并悬浮于浓度为10μM的低葡萄糖不完全DMEM中。菌落形成单位(CFU)/ml(来自LB培养基中约2ml对数期培养物的细菌细胞悬浮于1ml不完全DMEM中)。

  3. 细菌粘附于细胞系
    1. 使用在25cm 2烧瓶中生长至70-80%汇合的细胞系。
    2. V。将在低葡萄糖不完全DMEM中的霍乱弧菌培养物(200μl,约2×10 8 CFU)加入到70-80%汇合的细胞系中,得到的感染复数(MOI)约为50.
    3. 在添加细菌后,将细胞系在37℃下在5%CO 2加湿气氛中温育45分钟以获得最佳粘附。
    4. 45分钟后,弃去上清液,通过用5ml 1x PBS洗涤感染的细胞系4次除去未粘附的细菌,然后加入低葡萄糖完全DMEM(5ml)并孵育不同的时间(1- 12小时)。
    5. 在所需的时间后,弃去上清液,用5ml 1x PBS洗涤感染的细胞系4至5次。
    6. 加入1ml 1%Triton X-100,在室温下温和涡旋孵育2分钟,此时可以看到细胞系从烧瓶中裂解和分离。在1.5中收集总培养物 ml微量离心管,并进行强力吸液或涡旋。 Triton X-100处理裂解真核细胞,但对细菌细胞的活力没有影响(见注3)。
    7. 在室温下通过离心(5,000×g,5分钟)收集细菌细胞,并将沉淀悬浮在1ml 1×PBS中。
    8. 从该悬浮液中,将10μl加入990μl的0.9%盐水溶液中,通过连续稀释和铺板在LB琼脂平板上测定细菌CFU。 剩余的粘附细菌细胞可用于基因表达研究或其他目的。
    9. 在相同条件下用霍乱弧菌进行平行实验,但没有细胞系作为未粘附对照。

笔记

  1. 将细胞系储存在具有10%DMSO作为冷冻保护剂的液氮中。
  2. HT29分化
    1. 基本上如Huet等人(1987)所述进行HT29细胞的分化。
      1. 未分化的HT29细胞在25cm 2烧瓶中的高葡萄糖完全DMEM中生长至70-80%融合。
      2. 加入1ml含有0.5%胰蛋白酶和1mM EDTA的溶液, 在约5分钟后,将分离的细胞用于接种25cm 2烧瓶 不含补充有20mM葡萄糖和1mM葡萄糖的完全DMEM   mM半乳糖(第0天)
      3. 然后下面的日程表 随后逐渐增加半乳糖的浓度至5mM 并降低葡萄糖的浓度:(a)第1天,15mM葡萄糖加   1mM半乳糖; (b)第2天,10mM葡萄糖加3mM半乳糖; (c)天 3,5mM葡萄糖加4mM半乳糖; (d)第4天,5mM半乳糖。 的 在4天的时间内每天更换培养基。 一旦适应, 细胞在无葡萄糖的完全DMEM中生长几代 (5mM)。
    2. 碱性磷酸酶(AP)活性被估计为HT29分化的标志物(Luongo等人,2002)。
      1. 分化的和未分化的HT29细胞从中释放 通过用1ml胰蛋白酶(0.5%) - EDTA(1mM)处理, mM)溶液,用1ml冷的1×PBS洗涤3次,并在冰上裂解 用1x细胞裂解缓冲液。
      2. 对于AP活性测定,50μl细胞 裂解物与1ml< 磷酸对硝基苯酯(1mg/ml) 测定缓冲液在室温下30分钟,并停止反应   通过加入100μl的0.1M K 2 HPO 4。
      3. 计算AP活性   作为在405nm(A <405)/mg总细胞蛋白质的吸光度。 针对没有底物温育的裂解物测量A 405。
      4. 总蛋白通过Bradford方法使用Bio-Rad蛋白测定染料试剂估计。
  3. 为了检查1%Triton X-100处理是否对细菌细胞有任何影响,将细菌培养物(OD大约600)分成2等份,一份用Triton X-100处理 2分钟,测定经处理和未处理的培养物的CFU。 如果两种培养物的CFU相当,则可以认为1%Triton X-100处理对本研究中使用的细菌细胞的存活率没有影响。

食谱

  1. 完成DMEM(100 ml)
    1.0 g DMEM(低或高葡萄糖)
    0.37克碳酸氢钠 10ml NCS(在56℃水浴中热灭活30分钟) 用去离子水将体积调节至100ml 使用0.22μm过滤器单元过滤
    储存在4°C
  2. 不完全DMEM(100 ml)
    1.0g DMEM(低葡萄糖)
    0.37克碳酸氢钠 用去离子水将体积调节至100ml 使用0.22μm过滤器单元过滤
    储存在4°C
  3. 无葡萄糖完全DMEM(100ml)
    89ml液体DMEM(无葡萄糖)
    10ml热灭活NCS
    使用0.22μm过滤器单元过滤
    储存在4°C
  4. 10x PBS(500ml,pH7.4) 40.0g NaCl
    1.0克KCl
    6.07g Na 2 HPO 4
    1.0g KH 2 PO 4 sub/
    用去离子水将体积调节至500ml,如果需要,用NaOH调节pH值
    高压灭菌
  5. 1x PBS(50ml)
    将5ml在45ml去离子水中稀释的10x PBS稀释 使用0.22μm过滤器单元过滤
  6. 1x胰蛋白酶-EDTA(10ml) 1ml用9ml无菌1×PBS稀释的10×胰蛋白酶-EDTA 将1ml等分试样分散在离心管中 储存在4°C
  7. 1%Triton X-100(50ml) 0.5ml 100%Triton X稀释在49.5ml无菌1x PBS中
  8. 0.9%盐水溶液
    0.9g NaCl在100ml去离子水中的溶液 高压灭菌
  9. LB培养基(100ml) 1克细菌胰蛋白胨(1%)
    0.5克细菌酵母提取物(0.5%) 0.5克NaCl(0.5%) 用去离子水将体积调节至100ml,用NaOH将pH调节至8.6 高压灭菌
  10.   LB琼脂(100 ml)
    100ml LB培养基
    1.5克Bacto琼脂(1.5%) 用去离子水将体积调节至100ml,用NaOH将pH调节至7.4 高压灭菌
  11. 碱性磷酸酶测定缓冲液(10ml) 1ml 1M Tris/HCl(pH9.5)(终浓度100mM) 50μl1M MgCl 2(终浓度5mM) 用去离子水调节至10ml体积 高压灭菌

致谢

该协议改编自Huet等人(1987); Luongo等人(2002); 和Dey等人(2013)。

参考文献

  1. Dey,A.K.,Bhagat,A.and Chowdhury,R。(2013)。 宿主细胞接触诱导毒力因子和VieA(一种环状二-GMP磷酸二酯酶)的表达 > Vibrio 霍乱。 细菌 195:2004-2010。
  2. Huet,C.,Sahuquillo-Merino,C.,Coudrier,E。和Louvard,D。(1987)。 从多能肠细胞系(HT-29)分离的吸收和分泌粘液的亚克隆提供了新的模型 用于细胞极性和末端分化。细胞生物学 105:345-357
  3. Luongo,D.,Mazzarella,G.,Della,R.F.,Maurano,F.and Rossi,M。(2002)。 在肠细胞系HT-29分化过程中ERK1和ERK2活性的下调。 a> Mol Cell Biochem 231:43-50。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dey, A. K., Bhagat, A. and Chowdhury, R. (2014). Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines. Bio-protocol 4(8): e1105. DOI: 10.21769/BioProtoc.1105.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。