The β-hexosaminidase activity (release) assay

The assay was performed essentially as described (107). HeLa (250,000), MDCK (800,000), and Caco-2_BBe (500,000) cells were seeded on a 6-well plate for 2 days, until reaching ~70% (HeLa), or full cell confluence (MDCK or Caco-2_BBe). Cells were infected with pre-activated EPEC for 60 min (HeLa) or 120 min (MDCK) at 37°C. EPEC was pre-activated in high glucose DMEM lacking phenol red (Biological Industries 01-053-1A). Extracellular media (~1.2 mL) were collected and centrifuged (1,000 × g, 3 min, 4°C), and the supernatant was placed on ice. The cells were washed three times with ice-cold PBS, lysed in 1% NP-40 (in PBS), centrifuged (11,000 × g, 5 min 4°C), and the detergent soluble fraction, i.e., cell lysate, was stored on ice. The β-hexosaminidase activity assay was applied to 100 µL of the medium. For determination of the cellular content of β-hexosaminidase, cell lysates were diluted 1:10 with PBS, and 100 µL of the diluted lysates was taken to determine the enzyme activity. The samples were incubated for 15 min at 37°C with 15 µL of 6 mM 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide substrate (Sigma, M2133) in sodium citrate-phosphate buffer, pH 4.5. The reaction was stopped by adding 30 µL of 2 M Na₂CO₃ and 1.1 M glycine, and the fluorescence was measured with a plate reader (Synergy H1 - Biotek - 1) at excitation 365 nm/emission 450 nm, gain 50. Results were expressed as enzyme activity measured in the cell medium (i.e., secreted enzyme) normalized to the enzyme activity measured in the cell medium and lysate (i.e., total enzyme activity).