PAR-1, PAR-2 and PAR-3 were amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s). PAR-4 was amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s). Beta-actin (β-actin) was used as positive control using the following primer sequences: PD-0332991 in vitro β-actin (sense) 5′-CCAAGGCCAACCGCGAGAAGATG-3′ and β-actin (antisense) 5′-AGGGTACATGGTGGTGCCGCCAG-3′; yielding a expected PCR product of 587 bp. Beta-actin was amplified
with 35 cycles (94 °C for 60 s, 60 °C for 90 s, 72 °C for 60 s). Negative control was performed for each reaction and included the omission of the reverse transcriptase or the omission of cDNA in the PCR mix. PCR products were resolved on a 1.5% agarose gel for visualization. Flow cytometry analysis was performed of the freshly isolated naïve CD14+ monocytes and the CD14+ monocytes cultured for 24 h with experimental conditions. Briefly, the freshly isolated naïve CD14+ monocyte cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide and subsequently used for incubation with fluorochrome-labelled antibodies. The CD14+ monocytes cultured with experimental
conditions for 24 h were placed on ice for 1 h. Subsequently, medium with CD14+ monocytes was transferred to 1.5-ml tubes and centrifuged at 900 g for 5 min at room temperature. Supernatants were harvested; the remaining CD14+ cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide, and centrifuged at 900 g for 5 min at room temperature. After centrifuging, Mirabegron freshly isolated naïve CD14+ monocytes as well as cultured CD14+ monocytes 3-deazaneplanocin A nmr were incubated with APC-conjugated monoclonal mouse anti-human CD14 antibody, PE-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F)
antibody, PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies for 30 min at 4 °C in the dark. After a final washing and centrifuging step, cells were fixated in 2% paraformaldehyde. All cells were analysed using the FACS Calibur (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA). For cytokine assays, naïve PBMCs and naïve CD14+ monocytes recuperated for 24 h and subsequently cultured according to the experimental conditions for 24 h were used. Supernatants were harvested, transferred to 1.5 ml tubes, centrifuged at 900 g for 5 min at room temperature and cryopreserved at −80 °C. Cytokine production (IL1-β, IL-6, IL-8, IL-10 and TNF-α) was determined in triplicate. Standard and positive control recovery for each ELISA assay was between 90–110%.