Sodium azide n3na

Poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) 70:30, Resomer LC 703 S, Mw 160 kDa, Tg 37 °C and poly lactide-co-glycolide (PLGA) 82:18, Resomer LG 824 S, Mw 33.4 kDa, Tg 54–60 °C were from Evonik Nutrition and Care (GmbH, 64275 Damstad, Germany). Vancomycin hydrochloride from Streptomyces orientalis (C₆₆H₇₅C_l2N₉O₂₄, HCl), Mw 1485.71 Da potency ≥ 900 µg per mg (as VMC base); Span^® 80; nonionic surfactant (sorbitan monooleate, sorbitan oleate); ammonium phosphate monobasic (NH₄H₂PO₄) analytical grade ≥ 98.0%, Mw 115.03 Da, 1.81 g/cm³; methanol (MeOH, CH₃OH) analytical grade ≥ 99.9%, Mw 32.04 Da; N,N-dimethylformamide (DMF, C₃H₇NO) analytical grade 99.8%, Mw 73.09 Da; phosphate-buffered saline tablet (PBS); phosphoric acid (H₃PO₄) analytical grade ≥ 85%, Mw 98 Da; and sodium azide (N₃Na) Mw 65.01 Da were from Sigma-Aldrich (Milano MI, Italy). Acetone (CH₃COCH₃), analytical grade 99.8%, Mw 58.01 Da; chloroform (CHL, CHCl₃) analytical grade 99.9%, Mw 119.38 Da; and dichloromethane (DCM, CH₂Cl₂), analytical grade 99.9%, Mw 84.93 Da, were from Carlo Erba (Carlo Erba SpA, Milano, Italy). Acetonitrile (CH₃CN) Mw 41.05 Da was from Merck (Darmstadt, Germany). In-house double-distilled water was filtered with 0.22 µm Millipore membrane filters before use (Millipore Corporation, Bedford, MA, USA).

We used pregnant mice on days 16.5 dpc when the placenta is fully mature and fetal-maternal interactions and exchanges are most active. The placentas were separated from extra-embryonic tissues, umbilical cord and fetus, cut in small pieces and gently crushed on nylon filter (Cell strainer Falcon; BD Biosciences, Ref. 352350) and then rinsed with ice-cold Phosphate Buffer Saline (PBS) (Gibco; Ref. 14190-094). The cell suspension was centrifuged at 280 g for 5 minutes. We then lysed red blood cells with ammonium-chloride lysis buffer for 1 minute and washed the cells with a high volume of Flow cytometry (FC) buffer composed of PBS-Fetal Calf Serum (FCS) 4% (Gibco; Ref. 10270-106)- Sodium azide (N₃Na) (Sigma-aldrich; Ref S2002-500G) 0.1% (FC buffer). After a 5 minutes centrifugation at 280 g, pelleted cells were resuspended in 80% Percoll (GE Healthcare Bio-Sciences AB, Ref. 17-0891-01). A 40% Percoll solution was then added delicately above the cell suspension and the gradient was centrifuged at 1510 g for 30 minutes (15°C) without break. Leukocyte-enriched cells recovered from the interface between the two Percoll layers were washed in a large volume of FC buffer, spun at 280 g 5 minutes and resuspended in FC buffer to be kept on ice and counted.