Figure 1.
Schematic representation of the isolation procedure of primary, bead-free endothelial cells (ECs).
(A) First, DSB-X biotin-labeled rat anti-mouse CD31-antibody binds to CD31-positive ECs. Subsequently, streptavidin-labeled Dynabeads bind to DSB-X biotin. After magnetic separation, the Dynabeads are removed by a biotin-streptavidin competition. (B) Separation of CD31-positive H5V cells mixed with CD31-negative CT26 tumor cells at a ratio of 20∶1. The proportion (%) of CD31-positive H5V cells and the mean fluorescence intensity (mfi) remained unchanged after a simulated digestion step. (C) After separation, the mean fluorescence intensity (mfi) remained stable after digestion (second bar), but dropped to 17% after bead separation (third bar). (D) The recovery rate of H5V cells after separation was greater 70%.
Figure 2.
Localization of necrosis/apoptosis-fraction and capillaries in heart and tumor (B16-F0, size between 0.3–0.4 cm3).
(A, C) H&E staining of heart and tumor, respectively. Arrows indicate the fraction of necrotic/apoptotic areas. (B, D) CD31 immunostaining of heart and tumor, respectively. Arrows indicate CD31-positive ECs (red).
Figure 3.
Weight of various tissues and counts of isolated ECs.
(A) Summary of the total weight of separate tissues; weight of heart (n = 10) and lung (n = 3) taken from 13–15 week-old female mice; weight of repair blastemas (n = 10) taken from a balloon which was induced by using Freund’s adjuvant/PBS-emulsion; weight of CT26 (n = 10) and B16-F0 (n = 10) tumors which were taken when the size was ranging between 0.3 and 0.4 cm3. (B) Corresponding isolated EC counts were calculated per 100 mg tissue.
Table 1.
Number of viable heart ECs, heart weight and EC adherence after two days as a function of mice age (n = 2).
Figure 4.
Size of primary ECs isolated from different tissues.
(A–C) Isolated ECs from heart, repair blastema and B16-F0 with bound Dynabeads. Arrows indicate specific binding of corresponding CD31-positive ECs to Dynabeads. (D–F) Isolated ECs from heart, repair blastema and B16-F0 after biotin-streptavidin competition without any bound Dynabeads. (G, H) Mean of the forward scatter (FSC) from CD31+ cells isolated from heart (n = 4), lung (n = 3), repair blastema (n = 2), CT26 (n = 4) and B16-F0 (n = 4) measured using flow cytometry. Asteriks represent significantly different values (p** ≤0.01; p*** ≤0.001).
Figure 5.
Identification and characterization of ECs derived from different origin.
(A) Identification of control EC line H5V and primary ECs isolated from heart, lung, repair blastema, CT26 and B16-F0 using flow cytometry with EC-specific antibodies directed against CD31, CD105, CD144, CD34, CD54 and CD102. (B, C) Proportion of positively stained cells and mean fluorescence intensity values (mfi) (CD31, CD61, CD105, CD144, CD34, CD54, CD102) from normal ECs (heart (n = 5), lung (n = 3)), repair blastema ECs (n = 2) and tumor ECs (CT26 (n = 3), B16-F0 (n = 3)). Asteriks represent significantly different values (p* ≤0.05; p** ≤0.01; p*** ≤0.001).
Table 2.
Mean fluorescence intensity (mfi) of CD31, CD105, CD144, CD34, CD54 and CD102 of heart ECs in dependency of the age of mice (n = 2).
Figure 6.
Differences in expression of EC markers derived from slow- and fast-growing tumors (CT26 and B16-F0).
(A, B) Growth curves and doubling times of CT26 (n = 15) and B16-F0 (n = 15), in vivo. Asteriks represent significantly different values (p* ≤0.05; p*** ≤0.001). (C) Mean fluorescence intensity values (CD31, CD105, CD144, CD34, CD54, CD102) from slow-growing tumor ECs (CT26 (n = 3)) and fast-growing tumor ECs (B16-F0 (n = 3)). Asteriks represent significantly different values (p* ≤0.05; p** ≤0.01).
Figure 7.
Identification of ECs by immunostaining.
(A–C) CD31 staining of H5V, primary heart and tumor ECs (B16-F0). (D–F) Isolectin B4-staining of H5V, primary heart and tumor ECs (B16-F0).
Figure 8.
Phenotypic and functional characteristics of primary heart and tumor ECs (B16-F0), in vitro.
(A, B) Adherent heart and tumor ECs after 2 days. (C, D) Adherent heart and tumor ECs after 7 days. (E, F) Heart and tumor ECs under flow conditions (4 ml/min) after 4 days. Before starting the flow cells were incubated for 2 days under static conditions and 1 day under low flow conditions (2 ml/min). Arrows indicate flow direction. (G, H) Vascular tube formation of heart and tumor ECs on matrigel. (I) Migratory capacity of heart and tumor ECs. Asteriks represent significantly different values (p** ≤0.01). (J, K) Number of total branching points and total loops of heart and tumor ECs on matrigel. Asteriks represent significantly different values (p* ≤0.05).