The PICF/GCF samples were eluted from paperstrips into 500 µl PBS by vortexing the sample vigorously for 30 sec, the paperstrip was removed and microorganisms in the sample were pelleted by centrifugation at 14 000 g for 10 min and the supernatant was transferred to a clean tube.
Measurement of RANKL, OPG, IL-1, IL-17A, IL-17E levels in PICF/GCF samples Commercial ELISA kits were purchased for the measurement of OPG (R&D Systems Abingdon, UK), sRANKL, IL-17A, IL-17E (Peprotech London, UK), IL-1 (Thermo Fisher Paisley, UK). The ELISA assays were carried out according to the manufacturers' recommendations. The optical densities were read at 450 nm with a background subtraction at 570 nm and the samples were compared with the standards. The minimum detection limits for the assays were as follows OPG: 7.56pg/ml; RANKL: 7.56 pg/ml; IL-17A, IL-17E: 0.79 pg/ml; IL-1: 0.79 pg/ml. The PICF/GCF data were expressed as total amounts per sampling time (pg/30sec) and also as concentrations (pg/µl).
Measurement of RANKL, OPG, IL-1, IL-17A, IL-17E levels in PICF/GCF samples Commercial ELISA kits were purchased for the measurement of OPG (R&D Systems Abingdon, UK), sRANKL, IL-17A, IL-17E (Peprotech London, UK), IL-1 (Thermo Fisher Paisley, UK). The ELISA assays were carried out according to the manufacturers' recommendations. The optical densities were read at 450 nm with a background subtraction at 570 nm and the samples were compared with the standards. The minimum detection limits for the assays were as follows OPG: 7.56pg/ml; RANKL: 7.56 pg/ml; IL-17A, IL-17E: 0.79 pg/ml; IL-1: 0.79 pg/ml. The PICF/GCF data were expressed as total amounts per sampling time (pg/30sec) and also as concentrations (pg/µl).