Leukocyte trasmigration through endoglin expressing transfectants. (A) Flow cytometry analysis of endoglin transfectants. Rat myoblast transfectants expressing human endoglin (L6E9-E) and the corresponding mock transfectants (L6E9-M) were analyzed by immunofluorescence flow cytometry using mAbs P4A4 (anti-endoglin), OX18 (anti-MHC class I antigen), as a positive control and X63 (left panels) as a negative control. (B-E) Transmigration assays. Transwell experiments were performed using 0.5-μm pore membranes previously covered with a confluent monolayer of myoblast transfectants (L6E9-M or L6E9-E). To test the migration rate, Nalm6, U937, THP-1, or K562 cells were seeded on top of the transwell and 100 ng/mL CXCL12 (unless stated otherwise) was added to the bottom of chambers. After incubation for 2 hours at 37°C, an aliquot (100 μL) of transmigrated cells was counted by flow cytometry. There is a significant increased transmigration through L6E9-E versus L6E9-M (B-E) that is clearly inhibited in the presence of soluble endoglin (B-C). The transmigration of K562 is much lower than that of Nalm6, U937, or THP-1 cells. Each bar represents the mean value of 6 (B) or 3 (C-D) assays each performed in triplicates (**P < .005; *P < .01; ns, not significant). (F) Confocal analysis of transwells after transmigration. K562 cells were labeled with CFSE, loaded in the wells and subjected to transmigration assays through L6E9-E or L6E9-M transfectants as in panels B and D. Then, remaining cells on transwells were visualized by confocal microscopy. CFSE-labeled cells emitting green light are on the left, monolayers of L6E9 transfectants and K562 transmigrating cells visualized by light microscopy are in the middle, and the merged figures are on the right. Magnification, ×200.