Lysophosphatidylcholine lpc

In order to investigate the contribution of the various cell components to the deformability in our microfluidic system, RBCs were incubated for 20 min at room temperature with 0.05% glutaraldehyde (GA), obtained by dilution of a 25% GA-stock solution (Sigma-Aldrich, St. Louis, MO, USA) with Ringer. Alternatively, treatment with lysophosphatidylcholine (LPC) (Sigma-Aldrich, St. Louis, MO, USA) consisted of incubation at room temperature at 10% hematocrit for five minutes with 5 to 10 μM LPC in Ringer.

Airway gene transfer was performed as described in Cmielewski et. al (2017) [26 (link)]. For all LV-dosing procedures, female C57Bl/6 mice (8–10 weeks of age at study commencement) were anaesthetised with an i.p. injection of a 10 µl/g body weight of a mixture of medetomidine (0.1 mg/ml, Orion Corp., Finland) and ketamine (7.6 mg/ml, Parnell Laboratories, Australia). Anaesthetised mice were non-surgically intubated with a 20 Ga i.v. catheter (BD Insyte, Becton Dickinson, USA), placed on a heating mat in a supine position and observed prior to any fluid delivery to ensure breathing was normal. The airways were conditioned with 10 µl of 0.1% lysophosphatidylcholine (LPC; Sigma-Aldrich) and one-hour later, 20 µl of the LV vector was delivered into the trachea in two 10 µl aliquots delivered ~60 s apart. The endotracheal tube was then removed, and anaesthesia was reversed with 2 µl/g body weight i.p. injection of atipamezole (0.5 mg/ml, Orion Corp., Finland). Animals were dosed according to the assigned dosing schedules outlined in Fig. 1 (n = 12 mice randomly assigned to each group). Sample sizes were determined based on previous studies.

Native lipoproteins were obtained from the human plasma of normolipidemic volunteers. The Ethics Committee approved the experimental protocol of the Faculty of Pharmacy of Universidad de Concepción, and all volunteers signed informed consent. Plasma samples were withdrawn using Ethylenediaminetetraacetic acid tetrasodium (EDTA, 0.5 mg/mL) (Sigma-Aldrich, St Louis, MO, USA) as an anticoagulant. LDLs were isolated by sequential potassium bromide density centrifugation. After removing chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein, the native LDLs were yielded at a final density of 1.019 to 1.063 g/mL [53 (link)].
Ox-LDL was obtained by exposing native LDL to CuSO4 (10 μM) for 4 h at 37 °C, getting a delta of absorbance at 234 nm of 0.4 units, as previously reported [53 (link)]. The oxidation process was ended with EDTA (2 mM) and Butylhydroxytoluene (BHT, 4.5 μM) (Sigma-Aldrich, St Louis, MO, USA). Ox-LDL was dialyzed with PBS, and LDL-protein concentration was quantified with a Protein DC kit (BioRad Laboratory, Hertfordshire, UK). Lysophosphatidylcholine (LPC, present in up to 40% of the total lipid content of ox-LDL) (Sigma-Aldrich, St Louis, MO, USA) was used in a parallel experiment measure its effect on Lox-1 in H4-II-E-C3 cells.