Cell imaging coverglasses

Cells were seeded over cell imaging cover glasses (Eppendorf). After the treatments, cells were washed in PBS and fixed in 4% paraformaldehyde in 0.1M phosphate buffer pH: 7.4 for 10 min at room temperature (RT). After fixation, cells were washed thrice in PBS, permeabilized and blocked with a solution containing 0.15% Tween-20 and 0.5% BSA for 30 min at RT. Once blocking solution was removed, and specific monoclonal antibody anti-α-tubulin (1:200) or mycotoxin phalloidin (1:500) was added and incubated overnight at 4 °C in a humid chamber. The next day, cells were washed in PBS and incubated with Goat anti-mouse fluorescent secondary antibody (Alexa Fluor 488, 1:500) for 1 h at RT. After incubation, cells were washed in PBS and the nuclei were stained with 4’, 6-diamino-2-phenylindole (DAPI) at a concentration of 1 μg/mL for 5 min at RT. Finally, the culture chamber was removed, and slides were mounted using Fluoromont G (Dako, Produktionsvej, Glostrup, Denmark). For the estimation of the globular (G): polymerized actin (F) ratio we carried out a protocol based on Deoxyribonuclease I kit (Invitrogen, Carlsbad, CA, USA) in combination with phalloidin staining.

Porcine endometrial luminal epithelial cells were isolated according to the method described by Blitek & Ziecik [28 (link)], with modifications. Briefly, uterine horns collected from gilts on days 11–12 of the estrus cycle (n = 4—7) were washed three times in sterile 0.01 M PBS (pH 7.4). Endometrium was separated from the myometrium and digested with 0.2% Dispase II (Sigma-Aldrich, USA) in HBSS (Ca-, Mg- and phenol red-free; pH 7.4; Sigma-Aldrich, USA) at room temperature for 45 min, using a magnetic stirrer. Released luminal epithelial cells were centrifuged at 200 × g for 8 min at 4 °C and washed in Medium 199 (M199; Sigma-Aldrich, USA) containing 10% Newborn Calf Serum (NCS, Sigma-Aldrich, USA) and 100 IU/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich, USA). Blood cells were removed using the Red Blood Cell Lysing Buffer (Sigma-Aldrich, USA). After final centrifugation, the obtained pellet was suspended in M199 containing 10% NCS and antibiotics and depending on the downstream analysis pleated onto 6-well plates (Corning, USA) or Cell Imaging Coverglasses (Eppendorf, Germany) and then cultured in M199 containing 10% NCS until reaching 90—100% confluence, before the beginning of a treatment.

To examine the presence of vWF (a marker of endothelial cells) and PTGIR proteins, pEETH and G1410 cells were cultured in Cell Imaging Coverglasses (0030742028; Eppendorf, Hamburg, Germany), fixed in 4% paraformaldehyde in PBS, and subjected to the staining procedure^{58 (link)}. The incubation with rabbit polyclonal anti-PTGIR receptor (10005518; Cayman Chemical; dilution 1:50) or rabbit polyclonal anti-vWF (A0082; Dako Cytomation, Glostrup, Denmark; dilution 1:50) antibodies was performed overnight at 4 °C. Negative control staining was accomplished by replacing the primary antibody with the rabbit IgG negative control (I-1000; Vector Laboratories, Inc., Burlingame, CA, USA). Afterwards, all sections were incubated with CY³-conjugated donkey anti-rabbit IgG (dilution 1:1000; 711-165-152; Jackson ImmunoResearch). Actin filaments and nuclei were visualized using CytoPainter Phalloidin-iFluor 488 Reagent (ab176753; abcam, Cambridge, UK) and diamidino-2-phenylindole (DAPI; Vector Laboratories), respectively. Slides were mounted in a Vectashield Mounting Medium (Vector Laboratories) and photographed using an Olympus digital camera.