Tecnai t12 tem microscope

The morphologies of PEG-poly(PDL-co-DO) NPs were analyzed using a Tecnai T12 TEM microscope (FEI, Hillsboro, OR). NP solutions (0.05 mg ml⁻¹, 10 μl) were placed on clean and hydrophilized CF400-CU TEM grids (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were stained with a 0.2% uranyl acetate solution for 15 s, washed three times in DI water, and then mounted for observation of NP images under the TEM microscope.

U87 and SF188 cells were plated at a density of 10,000 cells/well in 96 well plates. 24 h after, cells were treated with either fluorescent particles at a concentration of 100 µg/mL or left untreated as a control. At different time points cells were harvested, washed 3× and re-suspending cells in a cold 1% BSA solution on ice. Flow cytometry was performed using Attune NxT (Invitrogen) and at least 10,000 iterations were acquired, then the data was analyzed using FlowJo v.10.0.8r1. The mean fluorescence intensity (MFI) in the DiA channel was then recorded and divided by background MFIs from control cell populations for each time point to yield a normalized fold increase in MFI in this channel for each of the different cell types. As NPs from a singular batch preparation and thus with the same dye loading per weight ratio were used on all of these experiments, the normalized MFI can directly be translated to relative particle uptake.
For TEM, the same procedure was repeated as above except only in U87 cells plated at a density of 200,000 cells/well in 6 well plates. After harvest the cells were fixed in 4% paraformaldehyde then prepared for TEM imaging using a Tecnai T12 TEM microscope (FEI, Hillsboro, OR).