Abca3

hAE and hAMS cells were fixed immediately after isolation with 1% formaldehyde in PBS for 10 min. After two washing steps in 1% BSA (Sigma Aldrich, USA) in PBS, cells were either prior permeabilised for 20 min in Permeabilization Buffer (Affymetrix, USA) for intracellular staining or directly incubated with the respective primary antibodies for 45 min. Cells were washed and incubated with polyclonal Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor RPE-conjugated goat anti-mouse IgG secondary antibodies (1:750, Thermo Fisher Scientific, USA) for 30 min. Isotype controls were incubated with rabbit and mouse IgG. After washing, cells were analysed on a flow cytometer (Cytoflex, Beckman Coulter, USA). SP-A, Oct-4 and pan-CK were obtained from Santa Cruz Biotechnology (USA) and were used in a dilution of 1:100, all the other antibodies (SP-B, pro-SP-C, ABCA3, SSEA-4) were purchased from Abcam (UK) and were diluted 1:250. Every incubation step was performed on ice.