Ma5 28565

After 4% PFA fixation, cells were blocked by 5% bovine serum albumin (BSA, A7906, Sigma) in DPBS containing 0.1% Triton X-100 for 1 h, followed by the incubation of primary antibodies against integrin beta 1 (12G10, ab30349, Abcam, 1:200), paxillin (Y113, ab32048, Abcam, 1:200), CD49c (integrin alpha 3, ASC-1, MA5-28565, Invitrogen, 1:50), talin 1 (8D4, ab157808, Abcam, 1:100), vinculin (EPR8185, ab129002, Abcam, 1:100), FAK (#3285, Cell signaling Technology, 1:200), α-actinin (H-2, sc-17829, Santa Cruz Biotechnology, 1:200), and Phospho-Myosin Light Chain 2 (Thr18/Ser19) (pMLC, #3674, Cell Signaling Technology, 1:100) in 1% BSA for overnight at 4 °C. The samples were washed twice with PBS before applying fluorescence-conjugated secondary antibodies, including Goat Anti-Mouse lgG H&L (Alexa Fluor® 488) (ab150113, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 488) (ab150077, Abcam, 1:1000), Goat Anti-Rabbit lgG H&L (Alexa Fluor® 568) (ab175471, Abcam, 1:1000), Donkey Anti-Rabbit lgG H&L (Alexa Fluor® 647) (ab150075, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 568) (ab175473, Abcam, 1:1000), Goat Anti-Mouse lgG H&L (Alexa Fluor® 647) (ab150115, Abcam, 1:1000), in 1% BSA with or without dye-conjugated phalloidin and DAPI (R37606, Invitrogen) at room temperature for 45 min.

Cell monolayers were digested to obtain single-cell suspensions. Antibodies against integrin beta 1 (12G10, ab30394, Abcam, 1:200), integrin alpha 3 (ASC-1, #MA5-28565, Invitrogen, 1:100) or Isotype controls (mouse IgG, ab37355, Abcam, 1:100) was added to the single cell suspensions and incubated the cell suspensions on ice for 30 min. Cell suspensions were then washed with cold DPBS twice for 10 min before incubated with the Alexa Fluor488-conjugated secondary antibody (mouse IgG, ab150113, Abcam, 1:1000) for another 30 min on ice. After washed with DPBS twice, the cell suspensions were loaded into Imaging Flow Cytometer (ImageStream X Mark II, Merck) to count the cell populations using Amins IDAS 6.0.