X22 alexa fluor 555

Antibody colocalization studies were performed by confocal microscopy analysis in HPB-ALL cells, as previously described [30 (link)]. In brief, cells were incubated with B12 for 30–45 min, at 4 °C, washed, re-suspended in PBS and incubated with secondary anti-human IgG (H + L)-Alexa 647 antibody (Invitrogen) for 30 min at 4 °C. Cells were then carefully washed with ice-cold PBS and stimulated with or without 50 ng/ml IL-7 for 30 min at 37 °C. Cells were washed, fixed with ice-cold methanol for 2–3 min at − 20 °C, permeabilized and washed with PBS plus 0.05% tween, and incubated for 1 h at room temperature in permeabilization buffer with antibodies against: clathrin heavy-chain (X22-Alexa Fluor 555), EEA-1 (EPR4245-Alexa-647) and LAMP-2 (Alexa Fluor 555; all from ThermoFisher Scientific), followed by 4′,6-diamidino-2-phenylindole nuclear staining. Image acquisition was performed with the pinhole aperture set to 1 Airy Unit for the highest wavelength (633 nm) and adjusted for the lower wavelengths to maintain the same optical slice thickness for all channels. Up to 10 different fields of view with ~80 cells per field of view were collected for quantification. Percent c-localization was determined by counting the fraction of cells showing at least one colocalization event (minimum 3 × 3 pixels) between fluorophores from cells with intact nuclei and presenting both fluorophores.