Benzylpenicillin

Primary human trophoblast (PHT) cells were isolated from term placentas delivered by Caesarean section using a well-established method involving sequential trypsin digestion and Percoll purification [25 (link), 26 (link)]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO) and Ham’s F-12 nutrient mixture (Life Technologies, Carlsbad, CA) containing 10 % of fetal bovine serum (FBS, Atlanta Biological, Atlanta, GA), 50 μg/ml gentamicin, 60 μg/ml benzyl penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). Cells were plated at a density of 2.75 million in 35 mm dishes for subsequent protein analyses or 0.6 million per well in 12-well plates for uptake assays, and incubated in a 5 % CO₂ humidified atmosphere at 37 °C. The cells were cultured for a total of 90 h with daily change of the culture media. To confirm cells differentiation, release of chorionic gonadotropin (hCG) into the cell culture media was assessed with a commercial ELISA (Immuno-Biological Laboratories, Minneapolis, MN) at 18, 66, 90 h after plating the cells. hCG concentration in the culture media was 4.3-fold (66 h) and 7-fold (90 h) higher than the concentration at 18 h after plating (N = 4, P < 0.01; data not shown). All studies were repeated in PHT cells from 3 to 4 different placentas.

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Rockville, MD, USA). Lanthanum nitrate hexahydrate (purity 99.999%), 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), benzylpenicillin, streptomycin, dexamethasone, ascorbic acid, β-glycerophosphate, and alizarin red S (ARS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). An alkaline phosphatase (ALP) activity kit and a Coomassie brilliant blue protein assay kit were obtained from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).

Supernatants were generated by seeding 5 × 10⁴ cells per well into 48-well plates in 500 μL of DMEM/1% bovine serum albumin (BSA) containing 2 mmol/L L-glutamine, 60 μg/mL benzylpenicillin and 100 μg/mL streptomycin (all purchased from Sigma Aldrich, St. Louis, MO, USA). The conditioned supernatants were examined using a PGE2 enzyme-linked immunosorbent assay (ELISA, R&D Systems, Abingdon, UK).

Only analytical grade chemicals were used in this study. Acetonitrile, ethyl alcohol, formic acid, catechin, Folin-Ciocalteu reagent and gallic acid were purchased from Merck (Darmstadt, Germany). Apigenin, rutin, orientin, luteolin, kaempferol, quercetin, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Trolox, 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), phenanthroline, amikacin, gentamicin, benzylpenicillin, cefotaxime, fluconazole and ketoconazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibiotics were dissolved in sterile water. High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT with reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.

Fish meal was obtained from Pesquera Lota Protein Ltd. (Villagram, Chile). Casein and gelatin were obtained from Hulunbeier Sanyuan Milk Co., Ltd. (Inner Mongolia, China) and Rousselot Gelatin Co., Ltd. (Guangdong, China). Crystalline amino acids were obtained from Yimengsi Ltd. (Shanghai, China). L-Lysine, collagenase, dispase, insulin, transferrin, copper sulphate pentahydrate (CuSO₄·5H₂O), streptomycin sulphate, benzylpenicillin, triton X-100, dimethyl sulphoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Media (DMEM) were purchased from Hyclone (Logan, UT, USA). DMEM (lysine-free) was obtained from Beijing Tsing Skywing Bio. Tech. Co. Ltd. (Beijing, China).

Supernatants were generated by seeding 5 × 10⁴ cells per well into 48-well plates in 500 μL of RPMI-1640/1% bovine serum albumin (BSA) containing 2 mmol/L L-glutamine, 60 μg/mL benzylpenicillin and 100 μg/mL streptomycin (all purchased from Sigma Aldrich, St. Louis, MO, USA). The conditioned culture supernatants were collected and analyzed for the presence of PGE2 by specific ELISA kits according to the manufacturer’s instructions (R&D systems, Abingdon, UK).

1 × 10⁶ CFU/ml of bacterial strains were exposed to different concentrations of clindamycin, benzylpenicillin (both Sigma-Aldrich), LL-37 (Invivogen), Lysozyme (Sigma-Aldrich), HNP-1 (Peprotech), MPO, resistin, HBP (R&D Systems), and H₂O₂ (Sigma-Aldrich) for 3 h and plated in serial dilutions on casein agar plates. CFUs were analyzed after 24 h of incubation at 37 °C and 5% CO₂.