Nucblue live cell stain

Oligo libraries were denatured and linearized at 70 °C for 5 min and kept at 37 °C before hybridization. Cells were incubated with probes (10 nM final concentration) at 1 million cells/mL cell media for 1 h at 37 °C. Cells were then washed twice with 1x PBS, stained with a recommended concentration of antibodies at 4C for cell surface markers CD14 (monocytes), CD3 (T cells), CD19 (B cells), live/dead fixable yellow (BioLegend, cat. #301806 [RRID:AB_314188], 300406 [RRID:AB_314059], 302230 [RRID:AB_2073119] and 423104) and nucBlue live cell stain (Thermo Fisher cat. #R37605). For fluorophore only control Alexafluor 647 Isotype control was used (Biolegend cat. #400130). Cells were then washed twice with 1x PBS to remove excess.

Cells were seeded in 96-well plates (Cell Carrier™-96; PerkinElmer #6005550) at a density of 2 × 10³ cells per well. Nuclei were stained with NucBlue solution (NucBlue™ Live cell Stain, Thermo-Fisher Scientific #R37605) for 15 min at Room Temperature (25 °C) following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). After cell labelling for the nuclei, samples were washed three times in PBS and treated with DMSO, used as vehicle, and different MNPs@MIL[b] and MNP concentrations (5, 10, 15, 20 μg/mL). Cells were imaged using the PerkinElmer Operetta High-Content Imaging System (# HH12000000). Plates were read under confocal conditions using the 63× long WD objective. A specific fluorescence type channel was used to acquire images of NucBlue (Ex: UV light, Em: 460 nm) for nuclei staining, shown in blue. All images were analyzed using Harmony high-content imaging and analysis software (PerkinElmer, Waltham, MA, USA). Initial segmentation of cells was carried out in the DAPI channel by identifying the blue-stained nuclei with an area >30 µm. Finally, the number of spots per Area of Cytoplasm and Nucleus was expressed as mean per well.

The cell migration assay was performed using the OrisTM Cell 2-D migration of adherent cells assay kit (AMSBio, Cambridge, MA, USA), where plastic stoppers were used to simulate a wound [11 (link)]. Furthermore, the cells were dyed with NucBlue^® Live Cell Stain (Thermo Fisher Scientific Massachusetts, Waltham, MA, USA), which is a reagent that binds to DNA and can be excited by UV light at 360 nm, with an emission maximum at 460 nm. The stoppers were applied to a 96-well plate, where suspended dyed cells and the açaí extracts (50 μg/mL) were added to each well, except for the controls that were run at the same time. For the blank readings, only the medium was added to the wells; 10% of fetal bovine serum (FBS, Thermo Fisher Scientific Massachusetts, Waltham, MA, USA) was used as a positive control; and, for the full cell readings, the stoppers were never inserted to the wells, allowing the cells to migrate. The plate was incubated for 2 h and the fluorescence was read, and blank corrected, on the micro-plate reader SynergyH1 (BioTek, Winooski, VT, USA) at time zero. Moreover, the images of the cells were captured using the microscope EVOS FL Cell Imaging System (Thermo Fisher Scientific Massachusetts, Waltham, MA, USA). The cells were further incubated for another 48 h and the migration of the cells were evaluated both via florescence and imaging.

Transduced Pan02 cells were seeded at 1 x 10⁵ cells per mL in a 12 well plate in complete media +/- 10 ng/mL TNF (PeproTech) and incubated overnight. Twenty-four hours later, cells were stained using NucBlue Live Cell Stain (Thermo Fisher Scientific) according to manufacturer’s protocols. Several images per well were acquired on a fluorescent microscope (Invitrogen EVOS M5000) and automated counting of DAPI+ nuclei was used to determine the cell count in each image. Additionally, an MTT assay was then performed according to manufacturer’s protocols (Abcam). Transduced Pan02 cells were seeded at 1 x 10⁴ cells per well in a 96 well plate in complete media and incubated overnight. The following day, media was replaced with experimental media containing complete media +/- 10 ng/mL TNF (PeproTech) and allowed to incubate for 24 additional hours.