Fig. 5.
Fig. 5. Expression of TF in MP/HUVEC cocultures. (A) Endothelial cells cultured alone or endothelial cells coincubated with MP for 4 or 24 hours were treated thereafter with collagenase to prepare single-cell suspensions. For other samples, reverse-transmigrated MP were collected from the apical surface of intact cocultures (24-hour egressed MP). The latter did not require collagenase for removal. Aliquots of these suspensions were stained for flow cytometry using MoAbs against vascular cell adhesion molecule 1 (4B9; negative control for MP), cadherin 5 (hec 1; positive control for HUVEC), CD14 (3C10; positive control for MP, negative control for HUVEC), and TF (VD10). Dotted line profiles represent respective negative controls, solid lines indicate positive controls, and filled profiles correspond to cells stained for TF. MP and HUVEC were separated for analysis by gating on their distinct forward-scatter and side-scatter profiles. (B) The presence of TF in the cultures was also assessed by measuring PCA. Plots represent the mean ± SD of PCA in three to six individual 96-well cultures. Data are representative of four experiments. After incubation of monocytes with plain collagen gels or endothelial monolayers grown on collagen for 1 hour in the absence of added MoAb, cultures were washed to remove nonadherent lymphocytes. Then 20% FBS/M199 was added, with inclusion of LPS (1 ng/mL), VIC7 (20 μg/mL), or VD10 (20 μg/mL) in some samples. Individual cultures contained approximately 50,000 HUVEC on a 50-μL collagen gel and, when present, about 50,000 MP. Accordingly, PCA shown for unstimulated PBMC represents the activity observed in 50,000 peripheral blood monocytes. At 24 hours, PCA derived from cells remaining in the collagen gel was assessed separately from PCA in reverse-transmigrated MP collected from the same cultures. PCA from reverse-transmigrated MP is indicated by the stippled portion of the bar. For some samples, the SD was too low to be visible in the constructed graphs. PCA detected in HUVEC cultured alone was statistically increased over PCA in mock cultures of collagen gels lacking cells (collagen was coated with fibronectin and incubated in 20% FBS/M199 in similar fashion as the other cultures), P < .005. PCA detected in 4-hour and 24-hour cocultures of MP and HUVEC in the absence of added MoAb was significantly greater than the activity in either HUVEC alone or unstimulated PBMC, P < .005.

Expression of TF in MP/HUVEC cocultures. (A) Endothelial cells cultured alone or endothelial cells coincubated with MP for 4 or 24 hours were treated thereafter with collagenase to prepare single-cell suspensions. For other samples, reverse-transmigrated MP were collected from the apical surface of intact cocultures (24-hour egressed MP). The latter did not require collagenase for removal. Aliquots of these suspensions were stained for flow cytometry using MoAbs against vascular cell adhesion molecule 1 (4B9; negative control for MP), cadherin 5 (hec 1; positive control for HUVEC), CD14 (3C10; positive control for MP, negative control for HUVEC), and TF (VD10). Dotted line profiles represent respective negative controls, solid lines indicate positive controls, and filled profiles correspond to cells stained for TF. MP and HUVEC were separated for analysis by gating on their distinct forward-scatter and side-scatter profiles. (B) The presence of TF in the cultures was also assessed by measuring PCA. Plots represent the mean ± SD of PCA in three to six individual 96-well cultures. Data are representative of four experiments. After incubation of monocytes with plain collagen gels or endothelial monolayers grown on collagen for 1 hour in the absence of added MoAb, cultures were washed to remove nonadherent lymphocytes. Then 20% FBS/M199 was added, with inclusion of LPS (1 ng/mL), VIC7 (20 μg/mL), or VD10 (20 μg/mL) in some samples. Individual cultures contained approximately 50,000 HUVEC on a 50-μL collagen gel and, when present, about 50,000 MP. Accordingly, PCA shown for unstimulated PBMC represents the activity observed in 50,000 peripheral blood monocytes. At 24 hours, PCA derived from cells remaining in the collagen gel was assessed separately from PCA in reverse-transmigrated MP collected from the same cultures. PCA from reverse-transmigrated MP is indicated by the stippled portion of the bar. For some samples, the SD was too low to be visible in the constructed graphs. PCA detected in HUVEC cultured alone was statistically increased over PCA in mock cultures of collagen gels lacking cells (collagen was coated with fibronectin and incubated in 20% FBS/M199 in similar fashion as the other cultures), P < .005. PCA detected in 4-hour and 24-hour cocultures of MP and HUVEC in the absence of added MoAb was significantly greater than the activity in either HUVEC alone or unstimulated PBMC, P < .005.

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