Novocyte 3000 flow cytometer

All cells were suspended and fixed in 4% PFA as described above and then analyzed on a Novocyte 3000 Flow Cytometer and BD LSRFortessa (BD Biosciences, San Jose, CA). The median value of corresponding fluorescence intensity of all gated live single cells was used to evaluate the internalization of related NPs or macromolecules.

Details of the antibodies used and gating strategies for the identification of cell populations are provided in the supplementary tables 1 and 2. Staining of immune cells for flow cytometry was performed using a mix of fluorophore-conjugated antibodies and either LD Fixable Aqua Dead Cell Stain or Zombie NIR™ dye (BioLegend) in PBS. Fc receptors were blocked with anti-mouse CD16/CD32 antibody (BioLegend) for mouse staining. For intracellular staining, cells were fixed with either 4% paraformaldehyde (Electron Microscopy Sciences) or Foxp3 Fix/Perm fixation buffer (Foxp3/Transcription Factor Staining Buffer Set, eBioscience), washed with a permeabilization buffer, then stained for intracellular antigens in a Foxp3 Fix/Perm permeabilization buffer (eBioscience). After this, cells were washed with a permeabilization buffer and resuspended in PBS for analysis by flow cytometry according to the manufacturer’s instructions. Data were acquired on a BD™ LSRFortessa™ flow cytometer (BD Biosciences) or a NovoCyte 3000 flow cytometer and analyzed with FlowJo software.