Milliplex mouse cytokine chemokine magnetic kit

Quantification of the levels of the murine cytokines IL-2, IL-6, MIP-1-alpha, and TNF-alpha in mouse plasma samples was performed using a custom Milliplex Mouse Cytokine/Chemokine Magnetic Kit (Millipore) according to the manufacturer’s recommendations. Wash steps were performed using a Bio-Plex II Wash Station (Bio-Rad), and the data were acquired using a Bio-Plex 200 System with high-throughput fluidics (Bio-Rad). The resultant data were analyzed using Bio-Plex Manager 6.1 software.

After sampling and weighing, the left and right salivary glands (C57BL/6 viral sialadenitis model) or half of the left salivary gland (MRL/lpr lupus model) were placed into 1 mL Tris 50 mM / NaCl 0.1 M / Triton 0.1% buffer containing 1% protease inhibitor cocktail (Sigma Aldrich) in a lysing Matrix D tube (MP Biomedicals). Samples were then homogenized on a FastPrep 24 5G (MP Biomedicals) and centrifuged for 10 min at 14,000 rpm at 4 °C (Eppendorf Centrifuge 5417R). Supernatants were used for cytokine/chemokine determination. A Milliplex Mouse Cytokine/Chemokine Magnetic kit (Millipore) was used to quantify the cytokine/chemokine concentration in salivary gland supernatants following the manufacturer’s protocol. Samples were run undiluted on a Luminex 200 system using Luminex xPONENt software. Results are expressed as picograms per salivary gland (mean of right and left salivary gland; C57BL/6 viral sialadenitis model) or as picograms normalized to the whole left salivary gland (MRL/lpr lupus model).