Flow Cytometric Quantification of Fecal Microbiota

Microbial load of the study cohort was measured as described previously^{7 (link)}. Briefly, 200–250 mg of frozen (−80 °C) fecal aliquots was diluted in saline solution (0.85% NaCl; VWR International) and filtered using a sterile syringe filter (a pore size of 5 µm; Sartorius Stedim Biotech). Next, 1 ml of the microbial cell suspension obtained was stained with 1 µl of SYBR Green I (1:100 dilution in DMSO; Thermo Fisher Scientific) and incubated for 15 min in the dark at 37 °C. The flow cytometry analysis was performed using a C6 Accuri flow cytometer (BD Biosciences) according to Prest et al.^{11 (link)}. Fluorescence events were monitored using the FL1 533/30-nm and FL3 > 670-nm optical detectors. The BD Accuri CFlow software was used to gate and separate the microbial fluorescence events on the FL1/FL3 density plot from the fecal sample background. A threshold value of 2,000 was applied on the FL1 channel. Based on the exact weight of the aliquots analyzed, cell counts were converted to microbial loads per gram of fecal material.