The concentration of the X-gal product was determined via measuring the substrate concentration using the molar ratio (2:1 substrate to product). First, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, Biotium) underwent exhaustive enzymatic digest (15 units/mL) in phosphate buffered saline (PBS) containing 10 mM MgCl2 and 0.1 mM 2-mercaptoethanol [32 ,33 (link)]. After digest, there was a 24 -h wait time for oxidation and dimerization into the blue colored X-gal product dye (Fig. 1 B). A BioMate™ 3S Spectrophotometer (Thermo Scientific™, USA) was then used to measure the absorption spectrum of X-gal product, at 5, 50, or 500 μM dissolved in PBS, in the range of 500 nm–900 nm.
X gal
Manufactured by Biotium
X-gal is a colorimetric substrate used in molecular biology experiments. It is commonly used to detect the presence of beta-galactosidase activity in cells or organisms.
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2 protocols using x gal
1
Quantitative Determination of X-gal Concentration
2
Senescent Cell Detection Protocol
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For detection of senescent cells, 105 cells were plated per well in a 6-well plate in DMEM-C. After one day, the cells were fixed using 4% paraformaldehyde (PFA) for 5 min, rinsed using PBS and stained with 2 ml/well filtered (0.22 μm; Millex-GP) staining solution consisting of 36 mM citric acid, pH 6.0 (Sigma Aldrich), 4.5 mM potassium ferricyanide (Merck), 0.14 M NaCl, 1.8 mM MgCl2 (Merck) and 1 mg/ml X-gal (Biotium). After 6 h, the cells were imaged using a Primovert inverted microscope (Zeiss).
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