Cytation 3 plate reader

Plate reader assays were carried out on a Biotek Cytation III plate reader.
All confocal imaging experiments except those involving U2OS cells were carried out on a Zeiss LSM710 confocal microscope (Cornell Biotechnology Imaging Core Facility), equipped with an Inverted Axio Observer.Z1 microscope with 405, 458, 488, 514, 543, 561, and 633 nm laser lines. All U2OS cell-imaging experiments were performed on a Zeiss LSM700 confocal microscope (EPFL Bioimaging and Optics Platform), equipped with an Inverted Axio Observer.Z1 microscope with 405, 488, 555, and 639 nm laser lines.

The amount of protein present in the purified cell membrane material and cell membrane-coated NP was extracted using RIPA lysis buffer (phenylmethylsulfonyl fluoride, sodium orthovanadate, protease inhibitor cocktail, and 1x lysis buffer) and determined using a NanoOrange^® Protein Quantification Kit. To extract the protein, cells were trypsinized and washed with ice cold PBS. RIPA lysis buffer was added to cells and CMCNP at a concentration of 1 ml/ 1×10⁷ cells and 1 ml/mg CMCNP, respectively. Samples were agitated for 30 minutes at 4°C followed by a centrifugation at 19,802 ×g at 4°C. The supernatant was collected and stored at −80°C for further analysis. For quantification, samples were suspended in a 1x NanoOrange^® working solution at 0.5 mg/ml. The samples were incubated at 90–93°C for 10 minutes and cooled to room temperature for 20 minutes under light protected conditions, then 200 μl of the samples were added to a 96 well plate. Fluorescence intensity of the samples was measured using Cytation 3 plate reader (BioTek, Winooski, VT, USA) at excitation and emission wavelength of 485 nm and 590 nm, respectively. Western blot analysis was performed on the purified cell membrane material and cell membrane-coated NP with primary antibodies against CD47 and anti-α tubulin as the loading control.

Curcumin (CUR) and nanoparticle stability were evaluated using a Cytation 3 plate reader (BioTek, Winooski, VT, USA). NP and free CUR dissolved in DMSO (final DMSO concentration < 0.05 % v/v) were suspended in 1X PBS and RPMI media supplemented with 10% fetal bovine serum. 200 μl of 1 mg/ml NP solution was added to a 96 well plate. Fluorescence intensity (excitation/emission: 420/520) of the curcumin was recorded in every 30 minutes for 24 hours. Initial fluorescence intensity was considered as control, and the relative stability index was measured using the following equation:
$$\text{Relative}\text{stability}\text{index}=\frac{\text{Initial}\text{fluorescence}\text{intensity}}{\text{Fluorescence}\text{intensity}\text{at}\text{each}\text{time}\text{point}}$$