Evos light microscope 10x

DU145 and DU145R80 invasion assay was performed as previously described with minor modifications [63 (link)]. The role of ANXA1 on PCa cell invasiveness was analyzed in control conditions, by transfecting the cells with scrambled siRNAs or siANXA1s, by administration of an ANXA1 blocking antibody (AbANXA1) or control IgGRs, or by administration of nFPRs agonists/antagonists. The analysis of the effects produced on cell invasion was carried out after 48 h from treatments. Administration of nFPR agonists/antagonists to PCa cells was performed as follows: fMLP (50 nM), Ac2-26 (1 μM), Boc-1 (10 μM), ciclosporin H (CsH; 500 nM), WRW4 (10 μM). The number of cells that had migrated to the lower surface was counted in twelve random fields using EVOS light microscope (10X) (Life technologies Corporation). The experiments were performed in triplicate.

MIA PaCa-2 invasiveness was studied using the Trans-well Cell Culture (12 mm diameter, 8.0-fim pore size) purchased form Corning Incorporated (USA), as previously described19 (link). Briefly, at 24 hours after seeding or treatments, the Trans-well Cell Culture chambers were washed twice with PBS and fixed with 4% p-formaldehyde for 10 minutes, then with 100% methanol for 20 minutes. Following, the fixed cells were stained with crystal violet (0, 5% w/v in a v/v solution of 20% methanol/distilled water; Merck Chemicals) for 15 minutes. After that, the chambers were washed again in PBS and cleaned with a cotton bud to remove crystal violet exceedance. The number of cells that had migrated to the lower surface was counted in twelve random fields using EVOS light microscope (10X) (Life technologies Corporation).