Fig 1.
Morphology and growth rates of HUVECs and ECFCs.
(A) A sample of HUVECs seeded on gelatin coverslips. After 72 hrs. of culture with a seeding density of 30,000 cells/cm2, cells were fixed and stained with ProLong Mountant with DAPI and anti-VWF FITC antibody to visualize nuclei and VWF, respectively. (B) The same was done for ECFCs, showing WPB distribution. (C) Nuclei count of HUVECs and ECFCs were evaluated at varying incubation time points (4, 24, 48, 72, 96 hrs.) with a constant seeding density of 30,000 cells/cm2 (n = 3). The dotted lines represent the growth rate curves as analyzed by a linear regression line of each cell line. The growth rate slopes for HUVECs and ECFCs are 0.39 and 0.30, respectively. The slopes are not significantly different, and P-value shown was determined by linear regression (GraphPad Prism).
Fig 2.
Effect of incubation time and seeding density on WPB/Nuclei count for HUVECs and ECFCs.
(A) Evaluation of WPB/Nuclei count for both HUVECs and ECFCs in response to increasing incubation time (24, 48, 72, and 96 hrs.) with a constant seeding density of 30,000 cells/cm2 (n = 3). (B) The linear regression analysis of WPB/Nuclei count for a constant seeding density. (C) WPB/Nuclei count is evaluated in response to varying seeding densities (5,000, 10,000, and 30,000 cells/cm2) at a constant incubation time of 96 hrs. (n = 3). (D) The linear regression analysis of WPB/Nuclei count for a constant incubation time.
Fig 3.
Assessment of WPB/Nuclei count in sub-confluent versus confluent cultures.
WPB/Nuclei count is assessed in both sub-confluent and confluent cell cultures. Confluence was determined to be 51 cells/field for HUVECs and 75 cells/field for ECFCs as established in methods. A 1-way ANOVA was performed to analyze the WPB/Nuclei count of HUVECs and ECFCs in sub-confluent v. confluent cell cultures, ** p<0.0021, *** p<0.0002 (n = 3).
Fig 4.
Comparison WPB/Nuclei count depending on culture method.
WPB/nuclei count for constant incubation time (96 hrs.) and for constant seeding density (30,000 cells/cm2) are shown. A 1-way ANOVA was performed to analyze the WPB/nuclei between constant incubation time and constant seeding density for HUVECs and ECFCs, ns p>0.05 (n = 3).