The bacterial strains, their derivatives, and the plasmids used in this study are listed in Table 1 . Lactobacillus paracasei was grown in de Man, Rogosa, and Sharpe (MRS) (Merck GmbH, Darmstadt, Germany) medium at 30°C. Lactococcus lactis subsp. lactis and Enterococcus faecalis were grown at 30°C in M17 medium (Merck GmbH, Darmstadt, Germany) supplemented with 0.5% glucose (GM17). Staphylococcus aureus, Pseudomonas aeruginosa PAO1 and Escherichia coli DH5α and O157:H7 were grown in Luria-Bertani medium (LB) at 37°C with aeration. Erythromycin was added for a final concentration of 10 μg/ml or 300 μg/ml for lactic acid bacteria (LAB) and E. coli, respectively. Ampicillin was added for a final concentration of 100 μg/ml for E. coli. When necessary, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) (Fermentas, Vilnius, Lithuania) was added to LB plates for a final concentration of 40 μg/ml for blue/white color selection of colonies.
5 bromo 4 chloro 3 indolyl β d galactoside x gal
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania
5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) is a chromogenic substrate used in molecular biology and microbiology. It is used to detect the presence of β-galactosidase enzyme activity in bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
M17 medium, by Merck Group (2 mentions)
Citric acid, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Potassium ferricyanide, by Merck Group (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Potassium ferrocyanide, by Merck Group (2 mentions)
Sodium phosphate, by Merck Group (2 mentions)
6 protocols using 5 bromo 4 chloro 3 indolyl β d galactoside x gal
1
Bacterial Growth Conditions and Antibiotic Selection
2
Senescence-Associated β-Galactosidase Assay
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Cells were fixed for 15 min (room temperature) in 1% formaldehyde, washed with PBS and incubated at 37°C (no CO2) with fresh staining solution: 0.3 mg/mL of 5-bromo4-chloro-3-indolyl β-D-galactoside (X-Gal, Fermentas), 40 mM citric acid (Sigma), 40 mM sodium phosphate (Sigma) (stock solution (400 mM citric acid, 400 mM sodium phosphate) must be at pH6), 5 mM potassium ferrocyanide (Sigma), 5 mM potassium ferricyanide (Sigma), 150 mM NaCl (Sigma), 150 mM MgCl2 (Sigma). SA-β-GAL-positive cells were quantified after 16-20 hrs as compared to unstained cells.
3
Senescence-Associated β-Galactosidase Assay
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Cells (50,000 cells/6-well plates for 4 days of treatment) were fixed for 15 min (room temperature) in 2% formaldehyde, washed with PBS and incubated at 37 °C with fresh staining solution: 0.3 mg/ml of 5-bromo4-chloro-3-indolyl β-d -galactoside (X-Gal, Fermentas), 40 mM citric acid (Sigma), 40 mM sodium phosphate (Sigma; stock solution (400 mM citric acid, 400 mM sodium phosphate) must be at pH 6), 5 mM potassium ferrocyanide (Sigma), 5 mM potassium ferricyanide (Sigma), 150 mM NaCl (Sigma) and 150 mM MgCl2 (Sigma). The senescence-associated (SA)-β-Gal-positive cells were quantified after 16–20 h as compared to unstained cells.
4
Cultivation and Selection of Bacterial Strains
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The strains, their derivatives and plasmids used in this study are listed in Table 1 . L. paracasei was grown in De Man-Rogosa-Sharpe (MRS; Merck GmbH, Darmstadt, Germany) medium at 30°C. Lactococcus lactis subsp. lactis was grown at 30°C in M17 medium (Merck) supplemented with 0.5% glucose (GM17). Pseudomonas aeruginosa PAO1 and E. coli DH5α and M15 used for cloning and propagation of constructs were routinely grown in Luria-Bertani medium (LB) at 37°C with aeration. To obtain solid medium, agar (15 g/l; Torlak, Belgrade, Serbia) was added. Erythromycin was added to a final concentration of 10 μg/ml and 300 μg/ml for LAB and E. coli, respectively. Ampicillin and kanamycin were added to a final concentration of 100 μg/ml for E. coli. When necessary, 5-bromo-4-chloro-3-indolyl-β-D -galactoside (X-Gal; Fermentas, Vilnius, Lithuania) was added to LB medium plates at a final concentration of 40 μg/ml for blue/white color selection of colonies.
5
Embryonic X-Gal Staining Protocol
6
Embryonic X-Gal Staining Protocol
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Embryos were isolated female that is at day 15 of her pregnancy. Embryos were removed and placed in PBS and fixed by immersion in 4% PFA for a further 60 min. Embryos were incubated in staining solution containing 0.1% X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactoside (ThermoFisher) at 4°C for 48 h followed by a post-fixation with 4% PFA overnight at 4°C. Clearing of the tissues was performed in glycerol, and the resulting resource was stored in the dark at room temperature.
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