Cytation 5

Double-labeled immunofluorescence staining was performed on fixed and paraffin-embedded rabbit lung slides (5 µm) and on fixed rabbit empyema PF cultured on 8-well EZ slides (Millipore Sigma, Burlington, MA, USA) for 7 days. PAI-1, Fibrin(ogen), and nuclei were probed for using Alexa Fluor 488 (Jackson Immunoresearch, West Grove, PA, USA), Alexa Fluor 647 (Jackson Immunoresearch, West Grove, PA, USA), and Hoechst 33342 (Thermo Fisher, Waltham, MA, USA), respectively. Fluorescence imaging of the stained lung tissue slides were performed at 4× and 20× magnification using Cytation 5 (Biotek, Winooski, VT, USA). Z-stack imaging was performed on the stained 3D ex vivo culture slide at 40× magnification using Cytation 5 (Biotek, Winooski, VT, USA) as well.

Cell proliferation after a rescue experiment was obtained using the Cytation5 (BioTek) as a luminescence reader. Cells were plated at 10,000 cells/well at either 30 μg/ml, 50 μg/m of oleic acid (O3008, Sigma-Aldrich)-treated media, or plain media without the oleic acid. The oleic acid media was prepared by adding the oleic acid to the aliquots of media and then sonicating the solution for 60 s (duty 20, output 1). The proper number of cells were then spun down and resuspended with the either plain media or oleic acid media and plated in 96 white walled plates (Coring Incorporated Costar, 3610). Some wells were then treated 24 h later with either 20 μg/ml or 30 μg/ml of TOFA (Cay10005263-1000, Cayman Chemical Company). The plate was then read 24 h after TOFA treatment by adding 50 μL of CellTiter-Glo 2.0 (G9242, Promega) to each well to be read. The plate was then rocked on a shaker for 2 min and then taken off the shaker and incubated at room temperature for 10 more minutes. The plate was then read on the Cytation5 (BioTek) at 6 luminescence fibers at 135 nm for 1 s per well. Background values were subtracted (media + CellTiter-Glo) and average of 4 wells were normalized to vehicle-treated cells. Statistical significance was determined using Student’s t test.

Cell proliferation was obtained using the Cytation5 (BioTek) as a luminescence reader. Cells were plated at 3000 cells per well in 100 μL of media, and then let grow for 24 h in a 96-well white walled plate (Coring Incorporated Costar, 3610). After 24 h, cells were treated with TOFA (Cay10005263-1000, Cayman Chemical Company), Firsocostat (S8893, Selleck Chemicals), TBV-2640 (S9714, Selleck Chemicals), or CP-640186 (PZS0362, Millipore Sigma) at the following concentrations: .1, .3, 1, 3, 10, and 30 μg/ml. After either 24 or 48 h of growth under treatment, 50 μL of CellTiter-Glo 2.0 (G9242, Promega) is added to each well to be read. The plate was then rocked on a shaker for 2 min and then taken off the shaker and incubated at room temperature for 10 more minutes. The plate was then read on the Cytation5 (BioTek) at 6 luminescence fibers at 135 nm for 1 s per well.

The HepG‐2 tumor spheroids were formed and grown in 6‐well plates with ultra‐low attachment surface (Corning Incorporate) according to previous procedures.^[25] Briefly, 1 mL of HepG‐2 cells was seeded into each well at a density of 400 000 cells/well. After tumor spheroids with diameters up to 400 µm, the tumor spheroids were removed to 35‐mm confocal dishes. Then the NK cells were labeled with Cell Tracker Red CMTPX Dye and were visualized with red fluorescence. The NK cells (1 × 10⁶ cells) were pretreated with or without BPQDs@HSA (5 µg mL⁻¹) for 6 h, and then added into HepG‐2 tumor spheroids. The process of NK cells penetrated in tumor spheroids was determined by a cell imaging multi‐mode reader (Cytation 5, BioTek Instruments, Inc.). Furthermore, after NK cells were incubated with tumor spheroids for 12 h, the tumor spheroids were rinsed with PBS and scanned at the different layers from the top of the tumor spheroids to the middle using a confocal laser scanning fluorescent microscope. The video of HepG‐2 tumor spheroids attacked by BPQDs@HSA‐potentiated NK cells were determined by a cell imaging multi‐mode reader (Cytation 5, BioTek Instruments, Inc.).

Half-maximal inhibitory concentration (IC₅₀) values were obtained for MMTV-PyMT, MDA-MB-231, 4T1, and SUM159 cells by seeding 1000 cells/well in a 96-well plate for 24 h (h) and then treated with 0.01–500 µM of mebendazole (MBZ) or less than 1% dimethyl sulfoxide (DMSO) as vehicle control. After 48 h of treatment, PrestoBlue (Thermo Fisher) was added to achieve a 10% (v/v) concentration in each well, incubated for 4 h, and fluorescence was measured using a Cytation5 (BioTek Instruments). The IC₅₀ was calculated using a nonlinear fit log vs. response model. To calculate % cell survival, cells were fixed with 70% ethanol, stained with DAPI, and imaged using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective and DAPI filter. A 4 × 3 montage was used to capture the entire area of each well, and NIS Elements software (Nikon Instruments Inc.) was used to threshold the DAPI positive area of the image which is presented as % survival.