Ec plan neofluar 10 0.30 ph1 air objective

Wound healing and open-field migration assays were performed using a two-well culture insert (Ibidi). pHCAECs were resuspended in 70 μl of complete vascular growth medium and plated at a density of 1,100 cells per well into each reservoir of a culture insert (Ibidi) that adhered to the bottom of a six-well plate (Corning) and grown in standard cell culture conditions. After confluence is reached, the insert was removed using forceps, and the plate was rinsed with DPBS to remove cell debris. Migrating cells were cultured in complete vascular growth medium supplemented or not with 100 ng/ml rh TNFα. Time-lapse images were acquired, at 10-min intervals over 24 h, in standard cell culture conditions on an Axio Observer Z1 inverted motorized microscope (Zeiss) equipped with an incubation system for live-cell imaging (PeCon), using an EC Plan-Neofluar × 10/0.30 Ph1 air objective, Axiocam 503 mono camera, and ZEN 2 software (all from Zeiss). Phase contrast images were recorded at a location of the migration gap and sites of open-field migration. Cell movement was analyzed using the Manual Tracking plugin for ImageJ (NIH). To quantify the dynamics of cell migration, such as velocity or Euclidean distance and accumulated distance, the migration trajectories were then assessed with Chemotaxis and Migration Tool 2.0 (Ibidi).

Confluent cell migration assay was performed using six-well plates (Corning). After confluence is reached, the cells were cultured in complete vascular growth medium supplemented or not with 100 ng/ml rh TNFα. Time-lapse images were acquired, at 10-min intervals over 36 h in standard cell culture conditions on an Axio Observer Z1 inverted motorized microscope (Zeiss) equipped with an incubation system for live-cell imaging (PeCon), using an EC Plan-Neofluar × 10/0.30 Ph1 air objective, Axiocam 503 mono camera, and ZEN 2 software (all from Zeiss). Phase contrast images were recorded at a location of the migration gap and sites of open-field migration. Cell movement was analyzed using the Manual Tracking plugin for ImageJ (NIH). To quantify the dynamics of cell migration, such as velocity or Euclidean distance and accumulated distance, the migration trajectories were then assessed with Chemotaxis and Migration Tool 2.0 (Ibidi).