Extraction of Gut Microbiota from Fecal Samples

The study sample comprised 5 fecal samples from healthy donors. No donors reported any severe diseases in the last 6 months. Fresh fecal material was collected in a sterile container and immediately manipulated and homogenized within a maximum time span of 2 h. Nine millilitres of sterile NaCl 0.9% (w/v) was added to 1 g of sample, and the mixture was homogenized in a sterile bag, using a laboratory paddle blender (Stomacher Lab Blender 400, Seward Ltd. UK) for 1 min. Microbiota extraction was then performed following the protocol described by Hevia and coworkers^{42 (link)}. A solution of Nycodenz 80% (w/v) (Progen Biotechnik GmbH, Heidelberg, Denmark) was prepared in ultrapure water, and sterilized at 121 °C for 15 min. A volume of 3 mL of the diluted, homogenized fecal sample was placed on top of 1 mL of the Nycodenz solution, and centrifuged for 40 min at 4 °C (10,000×g, MLS-50 Swinging-Bucket Rotor, Beckman Coulter, Indianapolis, IN). The upper phase (soluble debris) was discarded after centrifugation, and the layer corresponding to the microbiota was collected, washed once and resuspended in 1 mL of FC buffer (1 × MACSQuant Running Buffer, MILTENYI BIOTEC, Germany).