Aria fluorescence activated cell sorter

Generation of MDCK cells expressing either EphB2 or ephrinB1 ligands was described elsewhere^{59 (link)}. Briefly, MDCK cells were infected with lentivirus carrying EphB2 or ephrinB1 complementary DNAs. The canine E-cadherin–GFP fusion construct (gift from J.W. Nelson) was inserted downstream of the cytomegalovirus promoter into the FUW lentiviral vector backbone. The E-cadherin–Cherry fusion construct was derived from the E-cadherin–GFP FUW plasmid. Lentiviral particles containing those cDNAS were produced to infect the EphB2-positive (E-cadherin–GFP) or ephrinB1-positive (E-cadherin–Cherry) populations.
MDCK cells expressing homogeneous levels of anti-EphB2-mcherry (ab)/GFP (protein) or anti-ephrinB-GFP (ab)/Cherry (protein) were selected using an ARIA fluorescence-activated cell sorter (BD).

MDCK EphB2/Ephrin expressing cells were infected with lentivirus carrying lifeact-mcherry or lifeact-CFP (gift from J. de Rooij) and FAC sorted using an ARIA fluorescence-activated cell sorter (BD).

Flow cytometry data was collected using the following instruments: 5
laser (355, 405, 488, 561, and 640 nm) Bio-Rad ZE5 cell analyzer, 4 laser
(405, 488, 561, and 640 nm) Bio-Rad ZE5 cell analyzer, 4 laser (405, 488,
561, 640) Beckman Coulter Cytoflex cell analyzer. FACS was performed using a
3-laser (405, 488, and 633 nm) BD Biosciences Aria fluorescence-activated
cell sorter.
Confocal microscopy data was collected using a Zeiss LSM 710 laser
scanning confocal microscope with a 40x oil-immersion objective lens.