3d suspension array system

Cells were plated in 96-well plates coated with monomeric type-1 collagen and grown for 5 days under indicated conditions. For short-term signaling experiments, cells were treated with indicated antibody or inhibitor treatment in 2% serum-containing media for 4 h prior to addition of growth factor. Following growth factor addition, cells were kept at 37°C for indicated length of time and then reaction was stopped with a fast rinse of 4°C PBS followed by addition of 4°C Luminex cell lysis buffer. Cells were lysed for 30 min at 4°C on an orbital shaker and centrifuged at 14000 g for 10 min. Ten microliter of lysate was used for each Luminex reaction with a total volume of 50 μl in a 384-well plate. Beads were incubated with lysate and assay buffer overnight at 4°C followed by washing and addition of secondary antibody for 30 min and PE for 15 min. Samples were washed and read on a Bio-plex 3D suspension array system Luminex reader. Long-term experiments utilizing GSK3a/b inhibitor CHIR99021 used a 10 nM concentration for five days. Short-term Luminex experiments utilized a concentration of 10 nM pretreatment for 1 h prior to EGF addition and signaling analysis.