Sample collection
Six GCF samples are selected, two in each of the following categories: shallow (PD ≤3 mm), intermediate (PD = 4–6 mm) and deep (PD ≥7 mm). The selected sites are located on non-contiguous interproximal dental faces and preferably distributed among the four quadrants. After removal of the supragingival biofilm with sterile cotton pellets, the sites are isolated with cotton rolls and gently dried with an air syringe to eliminate the possibility of contamination with saliva. GCF is collected by inserting standard paper strips (Periopaper, Oraflow Inc., Smithtown, NY, USA), approximately 2 mm into the periodontal pocket for 20 s. Strips visually contaminated with blood are discarded. The GCF volume is measured in a calibrated device (Periotron 8000, Proflow Inc., Amityville, NY, USA) and the readings are converted to an actual volume (microliter, μL) by reference to a standard curve. Strips from the six selected sites are immediately placed into separate microcentrifuge tubes and stored at − 80 °C for subsequent assays.
Processing of samples
The GCF samples will be evaluated for their content of 20 cytokines by Multiplex Bead Immunoassay (MAGPIX® System, Merck Millipore, Billerica, MA, USA): 75 uL of PBS will be placed in each microtube containing the individual samples and then they will be vortexed for 15 s and centrifuged for 5 min at 1500 × g for elution. Samples will be analyzed using a multi-analyte method by means of a 20-multiplex fluorescent bead-based immunoassay for 20 cytokines/chemokines (granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-γ (INF-γ), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, IL-23, CXCL11 (ITAC), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-3α, TNF-α) using commercially available kits (MILLIPLEX® MAP 384-Well High Sensitivity Human Cytokine Magnetic Bead Panel, EMD Millipore, Billerica, MA, USA) and a plate reader (MAGPIX® System, Merck Millipore, Billerica, MA, USA), according to the manufacturer’s recommendations. The amount of protein in each sample will be extrapolated from standards using appropriate software (Beadview EMD Millipore, Billerica, MA, USA). The minimum detectable concentration of GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, IL-23, ITAC, MIP-1α, MIP-1β, MIP-3α, TNF-α are 0.86 pg/ml, 0.37 pg/ml, 0.32 pg/ml, 0.26 pg/ml, 1.49 pg/ml, 0.31 pg/ml, 0.14 pg/ml, 0.38 pg/ml, 0.16 pg/ml, 0.98 pg/ml, 0.27 pg/ml, 0.16 pg/ml, 0.57 pg/ml, 0.19 pg/ml, 6.91 pg/ml, 1.06 pg/ml, 1.86 pg/ml, 0.71 pg/ml, 0.70 pg/ml and 0,24 pg/ml, respectively. The results will be reported as concentrations of cytokines/chemokines per volume of GCF (pg/μL).