The procedure was repeated with the infant’s head turned 90�� to the left and then to the right in order to virtually ensure sampling from the right and the left lung, respectively. The first collected fluid, reflecting tracheo-bronchial milieu, was sent for microbiological culture. The second aliquot consisted on average prompt delivery of 1.8 �� 0.5 mL (about 40% of the instilled volume) and was diluted with 0.9% saline up to 2 mL and centrifuged (3,000 rpm; 4��C; 10′). Cell-free supernatants were separated, immediately frozen at -80��C and thawed only once for the experiments. All samples were sterile and did not show visible blood contamination.sPLA2 assay and identification in BALFBALF supernatants were centrifuged (12,000 rpm; 10′ and then 3,500 rpm; 3′) through a membrane-filter with a molecular weight cutoff of 30 kDa (Amicon Ultra; Millipore, Billera, MA, USA), as previously published .
This aims at separating secretory and cytosolic phospholipases (which weigh approximately14 kDa and 80 kDa, respectively ). sPLA2 total activity was then measured with a non-radioactive method, as previously published . Coefficient of variation and detection range were <5% and 5-80 IU/mL, respectively.Different sPLA2 subtypes were identified by western blotting, as follows. BALF samples were lyophilized after trifluoroacetic acid treatment and 20 ��g of their proteins were resuspended in phosphate-buffered saline and dye-sample loading buffer (50 mM Tris-HCl, pH 6.7; 10% glycerol; 3% SDS; 1% ��-mercaptoethanol; 0.01% bromophenol blue).
Samples were then boiled (100��C; 10′), centrifuged (450g; 5′; 4��C) and then loaded into the gels. Electrophoresis was performed using 12% SDS polyacrilamide and protein bands were later transferred into PVDF membranes (Millipore – Billerica, MA, USA) by semi-dry transfer system (Bio-Rad -Hercules, CA, USA), using standard protocols. After gentle shacking of 4 h in blocking solution (5% non-fat dry milk, PBS, and 0.5% Tween 20), blots were first incubated with anti-sPLA2-IB (polyclonal), -V (monoclonal), or -X (polyclonal) antibodies (Santa-Cruz Biotechnology, Santa Cruz, CA, USA). Anti-sPLA2-IIA (monoclonal) antibody (Cayman Chemical, Ann Arbor, MI, USA) was also used. All these antibodies were specific for human phospholipases and were incubated in blocking solution (1:200; 2 h).
Every excess of primary antibody was then removed twice using wash solution for 10′ and immune-reactive bands were subsequently incubated with goat anti-mouse and anti-rabbit IgG-HRP secondary antibodies (1:2,000) in blocking solution for 1 h at room temperature. Blots were washed four times for 10′ (each time) in PBS and 0.05% Tween 20 solution and Cilengitide blotted proteins were revealed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol with maximum exposure time of 1′.