Quantitect gene expression assays

BMDM or THP1 cells were plated in six-well microculture plates (at 5 × 10⁶ cells/well), stimulated during 3 h with LPS or PMA, respectively, and were washed and stimulated with particles for 4 or 6 h. RNA was extracted using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) and particles were removed by centrifugation (10 000 g, 10 min, 4 °C). All primers were synthesised (Qiagen). The expression levels of A1, A2_A, A2_B, A3, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 and P2Y13 receptor mRNAs, relative to housekeeping 18S mRNA, and expression of ENT1 and ENT2 transporter mRNAs, relative to housekeeping β2m mRNA, were analysed using Quantitect gene expression assays (Qiagen). Reverse transcription was performed by SuperScript III Reverse Transcriptase (Fisher Invitrogen) according to the manufacturer's instructions for amplification. RT-PCR was performed starting from 500 ng of total RNA using a Stratagene Mx3005P real-time PCR system (Agilent Technologies, Massy, France). For all experiments, biological quadruplicates and technical triplicates were performed.

RNA was purified from lung homogenates by using Trizol extraction protocol. Reverse transcription of RNA into cDNA was carried out with Go script reverse kit (Promega). RT-PCR was performed with Fast SYBR Green master mix on an ARIA MX (Stratagene MX3005P, Agilent technologies). Primers were synthetized (Qiagen, Hilden, Germany). The expression of pro-IL-18 and Mmp12 mRNA, relative to housekeeping 18S mRNA, were analyzed using Quantitect gene expression assays (Qiagen). For all experiments, biological quadruplicate and technical triplicate were performed.