Cell culture insert

A wound in the endothelial cell layer is considered as an initial stimulus for the development of atherosclerotic plaques (response-to-injury model) [45] (link). To mimic this situation in an in vitro setup, HUVEC were cultivated in cell culture dishes containing cell culture inserts (ibidi, Martinsried, Germany), to create a defined wound area. Subsequently, cells were exposed to CSEaq under different flow conditions (plate-and-cone viscometer). All experiments were performed in a humidified environment with 5% CO₂ at 37 °C. Direction of flow was perpendicular to the wound in the cell layer. Each sample was accompanied by a static control without further treatment (static time-matched controls). The increasing closure of the wound was documented for a time period of 10 h and quantified using the Fiji image processing package [46] (link).

The scratch wound healing assay was performed using cell culture inserts (ibidi GmbH) according to the manufacturer’s instructions on uncoated tissue culture plastic. A detailed description of the procedure can be found in Additional file 7. The rate of cell migration was measured by quantifying the intensity translocation values for three independent biological replicates per condition using a selective mask filter (Slidebook).

Fibroblasts (1 × 10⁴) were seeded in Cell Culture-Inserts (ibidi, Bavaria, Germany) with 70 ml of culture medium containing 10% FBS overnight. After the Culture-Insert was removed, a wound was formed. The cells were cultured at 37 °C for 24 h with culture medium containing 10% FBS after being washed with PBS. The zones were photographed by an inverted microscope (Olympus, Tokyo, Japan) at 0, 6, 12, and 24 h after the Culture-Insert was removed.

Cell culture inserts (Ibidi, Germany) were used according to the manufacturer’s instructions. Briefly, the culture inserts were placed on tissue culture dishes and BMMs were cultured and differentiated into osteoclasts inside one of the insert chambers. Upon appearance of multinucleated osteoclasts, osteoblasts or MC3T3-E1 cells were added to the empty adjacent chamber. After 5–6 h of incubation allowing the cells to adhere to the tissue culture dish, the inserts were gently lifted using tweezers enabling the cells to migrate towards the osteoclasts. Delay in retraction and protrusions upon contact were determined by visual inspection. Initiation of retraction was identified as the time point when a protrusion in contact with osteoclasts began to contract. Initiation of protrusions were identified as the time point of formation of the persistent protrusions that enabled the cell to migrate away from the site of contact. Cell velocities were computed by measuring the displacement of the cell nuclei.

Cells (7×10⁵) were plated in 10 cm tissue culture plates overnight. Next day the cells were treated with the indicated drugs for 24 h. They were then trypsinized and replated in 24 well tissue culture plates containing cell culture inserts (Ibidi, Verona, WI). Next day the inserts were removed and the cells were washed with PBS and the media was replaced. The fine scratch created by the inserts was photographed at various time points and analyzed by TScratch software (CSELab, ETH Zurich, Switzerland).

Primary HSC at day 8 were trypsinized and plated within the cell culture inserts (Cat no# 80209, ibidi, Munich, Germany) placed in a 24 well plate (0.25 x 10⁶/chamber). When cells formed confluent tight monolayer, they were serum starved for 24 h to arrest them in G₀ phase_. Inserts were then carefully removed, resulting in a gap of approximately 500 nm between the two monolayers. Cells were washed twice with sterile PBS and then cultured in DMEM media (10% heat inactivated FBS, 1% PSF) with or without recombinant mouse C5a (10 ng/mL), mouse PDGF-BB (PDGF) (50 ng/mL), or PDGF and C5a. This concentration of C5a was chosen based on physiologically relevant concentrations observed in liver
[27 (link),18 (link)] and preliminary experiments testing a range of concentrations from 5 to 30 ng/mL C5a. Maximal stimulation of migration was observed at 10 ng/mL (data not shown). In the experiment with CCR2 (MCP-1 receptor) antagonist (227016, Millipore), 227016 (10 nM) was added to the cell media 30 min prior to treatment with PDGF or C5a. Images were captured using Olympus IX81 Microscope at 0, 10, and 24 h. Gap width and rate of wound healing was analyzed using Wimasis Image analysis software (Munich, Germany).