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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Biomaterials. 2018 Sep 14;185:174–193. doi: 10.1016/j.biomaterials.2018.09.018

Fig.3. Immunocytochemistry, transmission electron microscopy and flow cytometry.

Fig.3.

Subcellular localization of NRPV and EuCF-RPV particles are shown. (A) Representative images of MDM treated with NRPV-CF®633 (red) and Alexa-Fluor 488 secondary antibody against Rab5, Rab7, Rab11, Rab14, or LAMP-1 primaries (green). (B) Representative images of MDM treated with EuCF-RPV (green) and Alexa-Fluor 594 antibodies against Rab5, Rab7, Rab11, Rab14, and LAMP-1 (red). The yellow color (merged images) shows colocalization of particles and Rab proteins. Nuclei are stained blue with DAPI. Images were captured with 63X objective on a Zeiss LSM 710 confocal microscope. (C) TEM ultrastructural evaluation of macrophage particle uptake and subcellular distribution. Intracellular uptake of NRPV and EuCF-RPV particles are shown. EuCF nanocrystals are seen within the EuCF-RPV-treated cells as black punctate structures. NRPV accumulations appear as white crystals within membrane-bound endosomal compartments. Areas of interest are bordered with black dotted lines and are presented in corresponding high-resolution images (ii–iii) and illustrate particles within membrane-bound intracellular structures. (D) Flow cytometry results for control MDM (left panel) and MDM exposed to NRPV particles (right panels) are shown. MDM were cultured in 50 mL teflon flasks (5 million/flask) and treated with CF®633 labeled NRPV particles (right panels) at 25 μM and 50 μM concentrations for 8 hours. (i) Population of cells analyzed by scatter analysis plot of control MDM and NRPV treated MDM. Healthy cells (within the rectangle) can be easily distinguished from dead cells (outside the rectangle). This shows MDM remain viable even after particle treatment. (ii) Single cell gate, (iii) Live/dead MDM gated using propidium iodide at excitation/emission: UV Blue; 350/450 nm and (iv) CF®633-NRPV uptake in MDM gated by using CF®633 excitation/emission: 630/650 nm. Control MDM (left panel) showed no uptake, while MDM exposed to CF®633-NPRV particles exhibit 68.8% and 80.7% particle uptake at 25 μM and 50 μM concentrations, respectively.