HEK293 cells plated at low density on poly-D-lysine coated glass coverslips were cultured in DMEM. Five hours after plating, the medium was replaced with neuron plating medium, and dissociated E18 hippocampal cells (3 × 10
[4 (link)] cells/cm
[2 (link)]) were plated onto the HEK293 cells. On the fifth day of co-culture, the cells were transfected with rat neuroligin-1 cDNA and the GFP-encoding vector, using Lipofectamine 2000 transfection agent. Five hours after transfection, the medium was replaced with AChE-conditioned medium or AChE-free conditioned medium in the presence of 10 μM physostigmine. Two days after transfection of neuroligin-1, the cells were processed according to the procedures used for neurons (see above) and then incubated with anti-synapsin (Chemicon, 1:1000) at 4°C overnight. Following incubation with Cy3-conjugated secondary antibodies at 4°C for 1 hour, the coverslips were rinsed and then mounted for confocal microscopic examination.
[4 (link)] cells/cm
[2 (link)]) were plated onto the HEK293 cells. On the fifth day of co-culture, the cells were transfected with rat neuroligin-1 cDNA and the GFP-encoding vector, using Lipofectamine 2000 transfection agent. Five hours after transfection, the medium was replaced with AChE-conditioned medium or AChE-free conditioned medium in the presence of 10 μM physostigmine. Two days after transfection of neuroligin-1, the cells were processed according to the procedures used for neurons (see above) and then incubated with anti-synapsin (Chemicon, 1:1000) at 4°C overnight. Following incubation with Cy3-conjugated secondary antibodies at 4°C for 1 hour, the coverslips were rinsed and then mounted for confocal microscopic examination.