Flow cytometry. Neutrophil cell surface adhesion molecule expression was determined by flow cytometry. Isolated neutrophils (10 × 106/ml) were incubated in RPMI with anti-CD11b-AlexaFluor488 and anti-CD62L-PE or anti-CD11a-PE, for 30 min, 4 °C, protected from light. Subsequently, cells were washed with PBS and fixed with 1% paraformaldehyde until analysis. Cells were analysed at 488 nm on a FACScalibur (BD Biosciences, Heidelberg, Germany) and CellQuest Software was used for acquisition. Data were expressed as mean fluorescence intensities (MFI) and % of positive cells (% gated) compared to a negative isotype control. Real-time PCR. Extraction of mRNA
Tipifarnib mouse and synthesis of cDNA: For extraction of neutrophil RNA, neutrophils (5 × 106 cells minimum) were pelleted at 4800 g for 20 min and RNA extracted using TRIzol, according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA).
Complementary DNA (cDNA) was synthesized and verified as previously described . Amplification and quantification of gene expression: Synthetic oligonucleotide primers were designed to amplify cDNA for conserved regions of the CD62L, alpha subunit of CD11a and alpha subunit of CD11b (PrimerExpress™; Applied Biosystems, Foster City, CA, USA). For primer Cabozantinib in vitro sequences, see Table 1. Primers were synthesized by Invitrogen (São Paulo, Brazil) and ACTB and GAPDH were used as control genes. All samples were assayed in a 12 μl volume containing 5 ng cDNA, 6 μl SYBR Green Master Mix PCR (Applied Biosystems) and adhesion molecule gene primers as well as GAPDH and ACTB primers in 96-well reaction plate (StepOne Plus – Applied Biosystems). To confirm accuracy and reproducibility of real-time PCR, the intra-assay precision was calculated according Olopatadine to the equation: E(−1/slope) . The dissociation protocol was performed at the end of each run to check for non-specific amplification. Two replicas were run on the plate for each sample. Results were expressed as the arbitrary units (A.U.) of gene expression when compared with the
control genes. Measurement of serum sL-selectin, IL-8 and ENA-78. Peripheral blood was collected in glass tubes without anti-coagulant and serum separated by centrifugation and stored frozen (−80 °C) until ELISA. Serum sL-selectin, ENA-78 and IL-8 were determined by high sensitivity ELISA (R&D Systems, Minneapolis, MN, USA and BD Biosciences, San Jose, CA, USA, respectively), according to the manufacturers’ instructions. Statistical analysis. All data are expressed as means ± SEM. Differences between groups were evaluated by ANOVA followed by Bonferroni’s test or by the Kruskal–Wallis test followed by Dunns test, as appropriate, unless otherwise specified. A P-value of ≤0.05 was considered statistically significant.