Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria
采用荧光探针标记细胞研究乳酸菌中的胞内酸稳态
发布: 2015年01月20日第5卷第2期 DOI: 10.21769/BioProtoc.1380 浏览次数: 13231
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The authors used this protocol in:
Aug 2014
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Abstract
Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus casei (L. casei).
Keywords: Fluorescent probe (荧光探针)
Loading cell (负载细胞)
Ph measurement (pH值测定)
Lactic acid bacteria (乳酸菌)
Intracellular acid-base homeostasis (细胞内酸碱平衡)
Materials and Reagents
- Lactococcus lactis IL1403
- Enterococcus faecalis JH2-2
- Lactobacillus casei (ATCC, catalog number: 334 )
- 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) (acid form) (Life Technologies, Molecular Probes®)
- Luria-Bertani broth (Sigma-Aldrich, catalog number: L3147 )
- MRS broth (Sigma-Aldrich, catalog number: 69966 )
- Citrate (Sigma-Aldrich, catalog number: C7254 )
- Malic acid (Supelco, catalog number: 46940U )
- Pyruvate (Sigma-Aldrich, catalog number: P2256 )
- D-(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
- D-(+)-galactose (Sigma-Aldrich, catalog number: G0750 )
- Electrode filling solution (Orion, catalog number: 900011 )
- Na2HPO4 (Merck KGaA, catalog number: 1006559 )
- NaH2PO4 (Merck KGaA, catalog number: 106349 )
- HCl (Merck KGaA, catalog number: 100317 )
- NaOH (Merck KGaA, catalog number: 106462 )
- Triton X100 detergent (Merck KGaA, catalog number: 648466 )
- Valinomycin (Sigma-Aldrich, catalog number: V0627 )
- Nigericin (Sigma-Aldrich, catalog number: N7143 )
- BCECF stock solution (see Recipes)
- 50 mM potassium phosphate (KPi) buffer (see Recipes)
Equipment
- Stove Lab Tech (LIB-080M)
- Centrifuge for Eppendorf tubes (Eppendorf, model: 5418 )
- Centrifuge Sorvall ST 16R for centrifugation of Falcon tube under refrigerated conditions (Thermo Fisher Scientific)
- Fluorescence spectrophotometer (PerkinElmer, model: LS55 )
- Thermostatic bath (Lauda Alpha, model: RA8 )
- pH/MV meters (Orion, model: 420A )
- 4 M KCl saturated with AgCl for combination Ag/AgCl pH electrode (Orion, catalog number: 900011)
- 5 ml Quartz cuvette (optical path 1 cm)
- pH microelectrode (Horiba, model: 9669-10D )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mortera, P., Zuljan, F., Magni, C. and Alarcón, S. H. (2015). Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria. Bio-protocol 5(2): e1380. DOI: 10.21769/BioProtoc.1380.
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分类
微生物学 > 微生物新陈代谢 > 其它化合物
细胞生物学 > 基于细胞的分析方法 > 离子分析
细胞生物学 > 细胞新陈代谢 > 其它化合物
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