4 6 diamidino 2 phenylindole dihydrochloride dapi

Immunofluorescence analysis of HBMEC grown in chamber slides and HIBCPP cells cultivated in the inverted cell culture insert model was performed as previously described [29 (link)]. The following antibodies were used: primary antibodies: chicken anti-vimentin (BioLegend, San Diego, CA, USA), goat anti-Met (Abcam, Cambridge, UK), mouse anti-Ecad (BD, Franklin Lakes, NJ, USA), and rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA, USA); secondary antibodies: goat anti-chicken Alexa Fluor^® 594, donkey anti-goat Alexa Fluor^® 594, goat anti-mouse Alexa Fluor^® 594, chicken anti-rabbit Alexa Fluor^® 488, and donkey anti-rabbit Alexa Fluor^® 488 (all Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000 in PBS/1% BSA) (Merck, Darmstadt, Germany). Densitometric analysis of Western blot bands normalized to actin was performed using the ImageJ software 1.53e [48 (link)].

TARO cells transfected with pCheryy/P2A FLAG-Ly6H in cover glass were treated with or without 0.35 U/mL phosphatidylinositol-specific phospholipase C (PI-PLC; Thermo Fisher Scientific, Inc.) in OPTI-MEM (Thermo Fisher Scientific, Inc.) for 1 h at 37 °C, and fixed for 15 min with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer at pH 7.4, then rinsed three times with PBS. Thereafter, cells were blocked with the blocking buffer (5% normal donkey serum (NDS) in PBS) for 30 min. Primary antibodies (monoclonal anti-FLAG M2 antibody, 1:100 dilution;) were applied in the blocking buffer for 1 h. After two washes in PBS, incubation (1 h) with donkey Alexa Fluor 647-conjugated anti-mouse antibody in blocking buffer was performed. After washing with PBS, cells were incubated with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Merck Millipore) to visualise nuclei, washed again and post-fixed with 2% PFA in 0.1 M PB for 10 min. PFA was removed by three rinses with PBS, coverslips were mounted in Fluoremount-G (SouthernBiotech, Birmingham, AL, USA), and examined using an Olympus FV-1000 confocal system with a 60 × objective lens (N.A. = 1.35).

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and Na-salt (DSPE-PEG 2000) were purchased from Lipoid, Ludwigshafen, Germany. Baicalein was purchased from Haoxuan Biotech, Xi’an, China. Chloroform, methanol, and tert-butanol were purchased from Stanlab, Lublin, Poland. Sodium chloride, dimethylsulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) were purchased from ChemPur, Piekary Śląskie, Poland. Sepharose 4B CL, thiazolyl blue tetrazolium bromide, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride, and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Merck, Darmstadt Germany. The DiL stain (1,1ʹ-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) was purchased from Thermo Fisher, Waltham, USA. The RPMI-1640 and MEM-Alpha cell culture media were purchased from Lonza, Basil, Switzerland. Fetal bovine serum (FBS), GlutaMAX (L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and 100× antibiotic-antimycotic were purchased from BioWest, Nuaillé, France. Carbon films were purchased from Lacey Formvar/Cu grids, SPI Supplies, West Chester, PA, USA. Agarose was purchased from Roth, Karlsruhe, Germany. The CellTiter-Glo 3D Viability Assay and RealTime-Glo Annexin V Apoptosis and Necrosis Assay were purchased from Promega, Madison, WI, USA.