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Bacterial Fluorescent-dextran Diffusion Assay
细菌荧光右旋糖酐扩散分析   

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Abstract

Antimicrobial peptides are known to disrupt bacterial membranes allowing solutes to flow across the membrane in an unregulated manner resulting in death of the organism. Disrupting the bacterial membrane would thus perturb the cells osmotic balance resulting in an initial influx of the external aqueous buffer. We have designed an assay to investigate how antimicrobial peptide concentration affects the ability of fluorescently labelled dextran moieties of differing molecular weight and hydrodynamic radii to cross membranes of viable bacteria. This assay was used to show that diffusion of low and high molecular weight dextrans into bacteria was a function of antimicrobial peptide concentration (Sani et al., 2013).

Keywords: Bacterial membranes(细菌细胞膜), Antimicrobial peptides(抗菌肽), Fluorescent dye(荧光染料), Cell osmotic balance(细胞渗透平衡), Membrane diffusion(膜扩散)

Materials and Reagents

  1. Bacteria of interest [e.g. Staphylococcus aureus (S. aureus)] (Sani et al., 2013)
  2. Rhodamine-dextran (RD) of different molecular weights [e.g. 40 kDa RD molecular weight (RD-40) (Sigma-Aldrich, catalog number: 42874 )]
  3. Fluorescein-dextran (FD) of different molecular weights [e.g. 4.4 kDa FD (FD-4) (Sigma-Aldrich, catalog number: 46944 )]
  4. LIVE/DEAD® BacLightTM Bacterial Viability and Counting Kit (Life Technologies, catalog number: L34856 )
  5. Dulbecco’s Phosphate Buffered Saline (Dulbecco’s PBS) (Sigma-Aldrich, catalog number: D8537 )
  6. Peptide of interest (e.g. Maculatin 1.1) (Sani et al., 2013)
  7. Bacterial growth media [i.e., particular for the bacteria of interest e.g. S. aureus was grown as a batch culture in Luria broth (Thermo Fisher Scientific) (see Recipes)] (Sani et al., 2013)

Equipment

  1. Flow cytometer (e.g. Beckman Coulter, model: Quanta SC-MPL)
  2. 96 well plates (flat bottomed) (Thermo Fisher Scientific, Nunc®, catalog number: 12565210 )
  3. Transfer pipette

Procedure

  1. Stock preparation
    1. Prepare 3.2 mM solution of FD4 in Dulbecco’s PBS (12.8 mg FD4/ml) and 3.2 mM solution of RD-40 in Dulbecco’s PBS (128 mg RD 40/ml). Note that Dulbecco’s PBS is used as it has been found not to affect the membranes of a wide range of bacteria in this assay. 3.2 mM solutions have been optimized for this assay using S. aureus and may need slight optimization for other bacteria.
    2. Prepare 2.5 x 106 cells/ml solution of heat-killed (55 °C for 1.5 h) bacteria of interest in the bacterial growth media as a compensation control for non-specific FD4 and RD40 binding to the bacteria.
      Note: Bacteria were counted, throughout, using the Quanta SC-MPL and the LIVE/DEAD® BacLightTM Bacterial Viability and Counting Kit. But other methods of bacterial counting using Petroff Hausser counting chamber or optical density estimation from growth curves and CFUs have been used.
    3. Prepare a 2.5 x 106 cells/ml solution of viable bacteria of interest in chilled bacterial growth media.
      Note: harvesting and processing of the bacteria must be completed at 4 °C using media at 4 °C and the bacteria then stored for the assay at 4 °C.
    4. Prepare a stock solution of the peptide of interest (e.g. 1 mg/ml) in the bacterial growth media.
      Note: Peptide may need to be initially solvated in DMSO to prevent precipitation in the bacterial growth media. The final DMSO concentration in the peptide stock solution needs to be 10% v/v, this concentration will equate to 5% v/v of DMSO in the initial assay dilution which does not affect the assay.
    5. Bacterial media conditioned 96 well plates; add 250 μl of the bacteria media/well and allow to sit for 16 h, 4 °C. Remove the media (aspiration) immediately prior to the assay.

  2. Assay protocol
    Critical point: Many bacteria can actively transport dextran, thus to inhibit active transport of dextran the assay must be completed at 4 °C or on ice. All solutions after being prepared must be chilled to 4 °C and kept at 4 °C or on ice. The viable bacteria need 30-40 min at 4 °C to inhibit active transport; this can easily be achieved if the harvested bacteria are centrifuged at 4 °C and pre-chilled media is used to resuspend the bacteria at 2.5 x 106 cells/ml which are then kept at 4 °C or on ice.
    1. Prepare serial dilutions of the peptide in the media conditioned 96 well plates, e.g. 200 μl of the peptide stock solution is added to the first well and 100 μl is transferred to the second well containing 100 μl Dulbecco’s PBS and mixed (10-15 times, pipetting up/down). This transfer process in repeated from the second to the third well, etc., to form a double serial dilution.
      Note: Gentle pipetting is required to avoid bubble formation and using the same tip in one serial dilution (high concentration to low) avoids peptide adherence to the tip, if tips are changed per dilution step.
    2. Add 10 μl of the 3.2 mM stock solution of FD4 and 10 μl of the 3.2 mM stock solution of RD40 to each peptide dilution. Or add 20 μl of each if using only one dextran.
    3. Add 80 μl of the 2.5 x 106 cells/ml of the bacteria of interest; gently mix with the transfer pipette and incubate at 4 °C for 60-90 min.
      Note: Options at this point would be to have a series of the incubation times to look at the rate of dextran dye diffusion.
    4. Prepare non-specific dextran binding control: to three wells containing 100 μl Dulbecco’s PBS, 10 μl of the 3.2 mM stock solution of FD4 and 10 μl of the 3.2 mM stock solution of RD40, add 80 μl of the 2.5 x 106 cells/ml of the heat killed bacteria of interest and incubate at 4 °C for the equivalent time as in step B3 above.
    5. Prepare active transport/non-specific dextran binding control: To three wells containing 100 μl Dulbecco’s PBS, 10 μl of the 3.2 mM stock solution of FD4 and 10 μl of the 3.2 mM stock solution of RD40 add 80 μl of the 2.5 x 106 cells/ml of the viable bacteria of interest and incubate at 4 °C for the equivalent time as in step B3 above.
      Note: These controls must have no peptide in the wells.
    6. Set up flow cytometer:
      1. Quanta SC-MPL flow cytometer was used and green fluorescence of FD-4 positive cells (525 ± 20 nm band-pass filter, typically FL1 channel) and the red fluorescence of the RD-40 positive cells (575 ± 15 nm band-pass filter, typically FL3 channel) were measured on a logarithmic scale and analysed simultaneously with the electrical volume (EV) and side scatter (SS) using a 488 nm laser.
      2. Other flow cytometers could be used which use forward and side scatter to discriminate bacterial cells in logarithmic scale mode.
      3. Fluorescent analysis gates (FD4, FD4/RD40 and RD40) were set to contain 1% of background fluorescence (defined as the non-specific adherence of the dextran molecules to heat-killed S. aureus cells and/or S. aureus cells harvested and maintained at 4 °C).
    7. Analysis of dye diffusion. Each test sample was kept at 4 °C or on ice for the analysis and was completed over 1 min, which typically captured 14,000 ± 2,000 counts/sample.
    8. Positive FD4 and RD40 bacteria were determined as being above background fluorescence and FD4-positive, FD4/RD40-dual positive and RD40-positive bacteria were plotted against peptide concentration.

Recipes

  1. Luria broth
    10 g of tryptone/L
    5 g of yeast extract/L
    10 g NaCl
    pH 7.5

Acknowledgments

None.

References

  1. Sani, M. A., Whitwell, T. C., Gehman, J. D., Robins-Browne, R. M., Pantarat, N., Attard, T. J., Reynolds, E. C., O'Brien-Simpson, N. M. and Separovic, F. (2013). Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism. Antimicrob Agents Chemother 57(8): 3593-3600.

简介

已知抗微生物肽破坏细菌膜,允许溶质以不受调节的方式流过膜,导致生物体死亡。 破坏细菌膜将因此干扰细胞渗透平衡,导致外部水性缓冲液的初始流入。 我们设计了一种测定,以研究抗微生物肽浓度如何影响不同分子量和流体动力学半径的荧光标记的葡聚糖部分穿过活细菌的膜的能力。 该试验用于显示低分子量和高分子量葡聚糖在细菌中的扩散是抗微生物肽浓度的函数(Sani等人,2013)。

关键字:细菌细胞膜, 抗菌肽, 荧光染料, 细胞渗透平衡, 膜扩散

材料和试剂

  1. 感兴趣的细菌[例如 金黄色葡萄球菌(金黄色葡萄球菌)](Sani等人,2013) br />
  2. 不同分子量(例如40kDa RD分子量(RD-40)(Sigma-Aldrich,目录号:42874))的罗丹明葡聚糖(RD)]
  3. 不同分子量的荧光素 - 葡聚糖(FD)[例如4.4kDa FD(FD-4)(Sigma-Aldrich,目录号:46944)]
  4. LIVE/DEAD ® Bac Light TM 细菌活力和计数试剂盒(Life Technologies,目录号:L34856)
  5. Dulbecco's磷酸盐缓冲盐水(Dulbecco's PBS)(Sigma-Aldrich,目录号:D8537)
  6. 感兴趣的肽(例如, Maculatin 1.1)(Sani等人,2013)
  7. 细菌生长培养基[例如,,特别是对于感兴趣的细菌例如 金黄色葡萄球菌在Luria肉汤(Thermo Fisher Scientific)(参见Recipes)](Sani等人,2013)中作为分批培养物生长。

设备

  1. 流式细胞仪(例如,Beckman Coulter,型号:Quanta SC-MPL)
  2. 96孔板(平底)(Thermo Fisher Scientific,Nunc ,目录号:12565210)
  3. 转移移液器

程序

  1. 库存准备
    1. 制备FDD在Dulbecco's PBS(12.8mg FD4/ml)中的3.2mM溶液和 3.2mM RD-40在Dulbecco's PBS(128mg RD 40/ml)中的溶液。 注意 使用Dulbecco's PBS,因为已经发现它不影响膜 的广泛范围的细菌。 3.2 mM溶液已经 使用S优化该测定。 aureus ,可能需要轻微   优化其他细菌
    2. 制备2.5×10 6个细胞/ml热灭活(55℃,1.5小时)感兴趣的细菌溶液 在细菌生长培养基中作为非特异性的补偿对照  FD4和RD40与细菌结合。
      注意:计数细菌, 使用Quanta SC-MPL和LIVE/DEAD BacLight TM 细菌活力和计数试剂盒。但是其他细菌方法 使用Petroff Hausser计数室或光密度计数 已经使用了来自生长曲线和CFU的估计。
    3. 准备a  2.5×10 6个细胞/ml冷冻的感兴趣的活细菌的溶液 细菌生长培养基。
      注意:收获和加工细菌 必须在4°C使用介质在4°C和细菌完成 储存在4℃的测定。
    4. 在细菌生长培养基中制备感兴趣的肽的储备溶液(例如1mg/ml)。
      注意: 肽可能需要最初在DMSO中溶剂化以防止 在细菌生长培养基中沉淀。最终DMSO 肽原液中的浓度需要≤ <10%v/v,这个 浓度在初始测定中将等于 5%v/v的DMSO 稀释,不影响测定。
    5. 细菌培养基 条件96孔板;加入250微升的细菌培养基/孔和 允许静置16小时,4℃。立即取出介质(吸液) 。

  2. 测定程序
    关键点:许多细菌可主动转运葡聚糖,从而抑制葡聚糖的主动转运,测定必须在4℃或冰上完成。制备后的所有溶液必须冷却至4℃并保持在4℃或冰上。活菌在4℃需要30-40分钟以抑制主动转运;如果将收获的细菌在4℃下离心,并且使用预冷冻的培养基以2.5×10 6个细胞/细胞/ml,然后保存在4℃或冰上。
    1. 在处于96孔的培养基中制备肽的系列稀释液   板,例如,将200μl肽储备溶液加入到第一个中   并将100μl转移至含有100μl的第二个孔中 Dulbecco's PBS并混合(10-15次,向上/向下移液)。 此转移   从第二井到第三井重复的过程,等。,形成a 双系列稀释。
      注意:需要温和的移液以避免 气泡形成和使用相同的尖端在一个连续稀释(高 浓度降低)避免肽粘附到尖端,如果尖端 每次稀释步骤改变。
    2. 加入10μl的3.2 mM原液 FD4溶液和10μl3.2mM RD40储备溶液 肽稀释。 或者如果仅使用一种葡聚糖,则添加20μl
    3. 加入80μl2.5×10 6个细胞/ml的感兴趣的细菌; 轻轻混合移液管,在4℃孵育60-90分钟 注意:此时的选项将是一系列的孵育时间,以查看葡聚糖染料扩散的速度。
    4. 准备非特异性葡聚糖结合对照:三个孔 含有100μlDulbecco's PBS,10μl的3.2mM储备液 FD4和10μl的RD40的3.2mM储备溶液,加入80μl的2.5μl  x 10 6个细胞/ml的感兴趣的热灭活细菌,并在4℃下孵育  °C等于上述步骤B3的等效时间
    5. 准备 活性转运/非特异性葡聚糖结合对照:向三个孔 含有100μlDulbecco's PBS,10μl的3.2mM储备液 FD4和10μl的3.2mM RD40的储备溶液中加入80μl的2.5×  10 6个细胞/ml的感兴趣的活细菌,并在4℃下孵育 等于上述步骤B3中的等效时间。
      注意:这些控件在井中不能有肽。
    6. 设置流式细胞仪:
      1. 使用Quanta SC-MPL流式细胞仪 绿色荧光的FD-4阳性细胞(525±20 nm带通滤波器,   通常为FL1通道)和RD-40阳性的红色荧光 细胞(575±15nm带通滤波器,通常为FL3通道) 以对数标度测量并与其同时分析 电容量(EV)和侧向散射(SS)。
      2. 其他   可以使用使用前向和侧向散射的流式细胞仪 以对数尺度模式区分细菌细胞。
      3. 荧光灯 分析门(FD4,FD4/RD40和RD40)设置为包含1%的分析门 背景荧光(定义为非特异性粘附的 葡聚糖分子与热灭活的金黄色葡萄球菌细胞和/或S。 aureus 细胞 收获并保持在4℃)。
    7. 染料分析 扩散。 将每个测试样品保持在4℃或冰上用于分析 并在1分钟内完成,其通常捕获14,000±2,000 计数/样本。
    8. 阳性FD4和RD40细菌 被确定为高于背景荧光和FD4阳性, 绘制了FD4/RD40-双阳性和RD40阳性细菌 肽浓度。

食谱

  1. Luria肉汤
    10g胰蛋白酶/L
    5克酵母提取物/L
    10克NaCl
    pH 7.5

致谢

没有。

参考文献

  1. Sani,M.A.,Whitwell,T.C.,Gehman,J.D.,Robins-Browne,R.M.,Pantarat,N.,Attard,T.J.,Reynolds,E.C.,O'Brien-Simpson,N.M.和Separovic, Maculatin 1.1通过孔隙机制破坏了金黄色葡萄球菌脂质膜。 Antimicrob Agents Chemother 57(8):3593-3600。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:O’Brien-Simpson, N. M., Pantarat, N., Walsh, K. A., Reynolds, E. C., Sani, M. and Separovic, F. (2014). Bacterial Fluorescent-dextran Diffusion Assay. Bio-protocol 4(14): e1191. DOI: 10.21769/BioProtoc.1191.
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