Saliva samples were collected by expectoration and placed in a 10× protease inhibitor cocktail (cocktail set III; Calbiochem/EMD, Gibbstown, NJ, USA), and saliva was centrifuged for 5 minutes at 550 × g. Baseline oral C. albicans carriage was determined by plating the supernatant fraction of spun saliva in triplicate on yeast peptone dextrose plates with antibiotics (to suppress growth of oral bacteria) and C. albicans colony enumeration after incubation at 30°C for 48 hours. Salivary C. albicans killing was determined by incubating the salivary supernatant at 37°C with 1 × 106C. albicans cells (strain CAF2-1) (1:1, v/v) for 1 hour
[21 (link)]. C. albicans cells were plated in triplicate for colony enumeration. For β-defensin 2 (BD2) assessment, the supernatant was analyzed using a BD2 enzyme-linked immunosorbent assay kit in duplicate or triplicate as volume allowed (Phoenix Pharmaceuticals, Burlingame, CA, USA). BD2 concentrations were normalized to the total protein content of centrifuged saliva, which was measured by the bicinchoninic acid assay (BioRad, Hercules, CA, USA).
[21 (link)]. C. albicans cells were plated in triplicate for colony enumeration. For β-defensin 2 (BD2) assessment, the supernatant was analyzed using a BD2 enzyme-linked immunosorbent assay kit in duplicate or triplicate as volume allowed (Phoenix Pharmaceuticals, Burlingame, CA, USA). BD2 concentrations were normalized to the total protein content of centrifuged saliva, which was measured by the bicinchoninic acid assay (BioRad, Hercules, CA, USA).
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