Briefly, 1×106 cells were washed in ice cold PBS, suspended in 1 ml cold blocking buffer (PBS/1% FBS) that contained 1 ug 1st antibody (8G4 or migG), and incubated on ice for 1h with occasional agitation. Cells were centrifuged (500 × g for 5 min), washed 3× with cold PBS, incubated with 100 ng/ml 2nd antibody (Alexa-488 labeled donkey anti-mouse IgG, Life Technology), and washed 3× with cold PBS. Cell suspensions were analyzed with FACScan (Becton Dickinson, San Jose CA). 104 events were collected for each analysis.
HUVEC migration and Matrigel tube assays were performed as previously described [7 (link),8 (link)]. Antibody affinity was determined with a binding titration assay using HUVEC and flow cytometry [13 (link)], an approach that is useful for determining affinity with cell-surface antigens and desirable in our case because, lacking recombinant TM4SF1 protein, we could not perform ELISA.
HUVEC migration and Matrigel tube assays were performed as previously described [7 (link),8 (link)]. Antibody affinity was determined with a binding titration assay using HUVEC and flow cytometry [13 (link)], an approach that is useful for determining affinity with cell-surface antigens and desirable in our case because, lacking recombinant TM4SF1 protein, we could not perform ELISA.
