In vitro Endothelial Permeability assay
Endothelial monolayers are seeded in transwells. On the day of the experiment, the transwells are placed in a in a diffusion chamber (NaviCyte Scientific, San Diego, CA). The permeability of the monolayer to FITC-dextran 70 was determined according to the Fick equation.
Calcium measurements and calcium waves analysis in endothelial cells
Changes in intracellular calcium concentration ([Ca2+]i) were detected using the fluorescent calcium indicator, Fluo 4 (Life Technologies, OR, USA). Fluo 4 was uploaded by incubating the primary cultures of endothelial cells with 10 μM Fluo 4-acetoxymethyl ester (Fluo 4-AM) for 1 h at room temperature (~25°C), and time-lapse measurements of [Ca2+]i were started after 20 min of equilibration using an Olympus BX50 WI microscope and an intensified CCD camera (Retiga
Fast 1394, QImaging) controlled by the IPLab software (Scanalytics, Inc.). Figure: Time-lapse of intracellular calcium increase in green.
Connexin Hemichannel opening in endothelial cells and post-capillary venules
Connexin hemichannel opening may be studied by measuring hemichannel-permeable molecule uptake, using ethidium bromide (EtdBr), whose uptake is proportional to hemichannel opening and exposure time. Figure: Sequence of post-capillary venules image in brightfield and EtBr uptake (red dots).
Primary Cultures of Mesenteric Endothelial Cells
Microvascular endothelial cells were isolated from rat mesentery vessels. Briefly, after removing the blood from the vessels by perfusing a sterile physiological buffer solution containing a mixture of antibiotics and antimycotics (Anti-Anti solution, Gibco, Invitrogen, NY, USA), mesenteries were incubated in a physiological saline solution containing collagenase type I and BSA at 37°C. After 1 h, the collagenase/BSA solution was removed by two successive applications of cell media and centrifugation. Pelleted cells were resuspended in cell media. Nonadherent cells were removed 4 h later, and the remaining adherent endothelial cells were kept at 37°C in a 5% CO2-95% air atmosphere at nearly 100% relative humidity. Currently, I´m developing a similar technique to obtain primary cultures of mouse mesentery vessels, and it will be published in 2023.
Cell lines culture
EAhy296 and postcapillary venular endothelial cells (CVEC) were grown in DMEM supplemented with fetal bovine serum, L-glutamine, penicillin and streptomycin.
ECV-304 transfected with eNOS construct: ECV-GFPeNOS-G2A (eNOS anchored in the cytosol) (in the figure), ECV-GFPeNOS-CAAX (eNOS anchored in the membrane), were grown in DMEM supplemented with fetal bovine serum (Invitrogen), L-glutamine, penicillin, streptomycin and were additionally supplemented with geneticin (G418).
Human dermal microvascular endothelial cells (HMVEC) and human umbilical vein endothelial cells (HUVEC) were from Lonza (Walkersville, MD). Cells were grown in EGMTM -2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM
In vitro Endothelial Permeability assay
Endothelial monolayers are seeded in transwells. On the day of the experiment, the transwells are placed in a in a diffusion chamber (NaviCyte Scientific, San Diego, CA). The permeability of the monolayer to FITC-dextran 70 was determined according to the Fick equation.
Figure: Time-lapse of intracellular calcium increase in green.
Calcium measurements and calcium waves analysis in endothelial cells
Changes in intracellular calcium concentration ([Ca2+]i) were detected using the fluorescent calcium indicator, Fluo 4 (Life Technologies, OR, USA). Fluo 4 was uploaded by incubating the primary cultures of endothelial cells with 10 μM Fluo 4-acetoxymethyl ester (Fluo 4-AM) for 1 h at room temperature (~25°C), and time-lapse measurements of [Ca2+]i were started after 20 min of equilibration using an Olympus BX50 WI microscope and an intensified CCD camera (Retiga
Fast 1394, QImaging) controlled by the IPLab software (Scanalytics, Inc.).
Connexin Hemichannel opening in endothelial cells and post-capillary venules
Connexin hemichannel opening may be studied by measuring hemichannel-permeable molecule uptake, using ethidium bromide (EtdBr), whose uptake is proportional to hemichannel opening and exposure time.
Figure: Sequence of post-capillary venules image in brightfield and EtBr uptake (red dots).
Primary Cultures of Mesenteric Endothelial Cells
Microvascular endothelial cells were isolated from rat mesentery vessels. Briefly, after removing the blood from the vessels by perfusing a sterile physiological buffer solution containing a mixture of antibiotics and antimycotics (Anti-Anti solution, Gibco, Invitrogen, NY, USA), mesenteries were incubated in a physiological saline solution containing collagenase type I and BSA at 37°C. After 1 h, the collagenase/BSA solution was removed by two successive applications of cell media and centrifugation. Pelleted cells were resuspended in cell media. Nonadherent cells were removed 4 h later, and the remaining adherent endothelial cells were kept at 37°C in a 5% CO2-95% air atmosphere at nearly 100% relative humidity. Currently, I´m developing a similar technique to obtain primary cultures of mouse mesentery vessels, and it will be published in 2023.
Cell lines culture
EAhy296 and postcapillary venular endothelial cells (CVEC) were grown in DMEM supplemented with fetal bovine serum, L-glutamine, penicillin and streptomycin.
ECV-304 transfected with eNOS construct: ECV-GFPeNOS-G2A (eNOS anchored in the cytosol) (in the figure), ECV-GFPeNOS-CAAX (eNOS anchored in the membrane), were grown in DMEM supplemented with fetal bovine serum (Invitrogen), L-glutamine, penicillin, streptomycin and were additionally supplemented with geneticin (G418).
Human dermal microvascular endothelial cells (HMVEC) and human umbilical vein endothelial cells (HUVEC) were from Lonza (Walkersville, MD). Cells were grown in EGMTM -2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM