Ix50 inverted microscope

Measurement of the membrane dipole potential (MDP) was performed in μ-Slide I^0.2 Luer poly-L-lysine imaging chambers (Ibidi), as described previously (27 (link)). In brief, 5 μl of 1 mg/ml Di-8-ANEPPS (Thermo Fisher) in ethanol was added to 1 ml RBC suspension at a concentration of 3 × 10⁷ cells/ ml in DPBS/BSA, and c000000000ells were incubated at 37°C for 1 h. Subsequently, cells were washed three times in DPBS/BSA to remove excess dye and resuspended in 1 ml DPBS/BSA with or without CAT-8015 (500 ng/ml) for further incubation at 37°C for 1 h. Ratiometric fluorescence images were captured by an Olympus IX50 inverted microscope (Olympus Optical, Hamburg, Germany) equipped with a Plan-Neofluar 63 × /1.25 oil immersion objective, an AVT Stingray F-145B camera and Live Acquisition 2.2.0.8 software.

Cultured cells were fixed with 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation) at 27 °C for 10 min. Color development was performed using a TRAP staining kit (Cosmo Bio, Tokyo, Japan). Stained cells were immersed in PBS and observed using an IX50 inverted microscope (Olympus, Tokyo, Japan) with a 10× objective and bright field (BF) filter at 27 °C. Images were acquired with an Advancam-HD-2 s camera (Advan Vision, Tokyo, Japan).

Antibodies used in immunofluorescence analyses were as described above for immunoblotting. Highly cross-absorbed Alexa Fluor anti-mouse 488, anti-mouse 594 or anti-rabbit 594 secondary antibodies were used for detection (Invitrogen). Image acquisition and quantification of BiFC signals were as described in^{17 (link)}. For Oil red O analysis of neutral lipid accumulation in adipocytes, following fixation, cells were treated with Oil Red O 0.1% solution further diluted in dH2O (6 parts Oil Red O 0.1% in 4 parts dH2O). Images were taken at Olympus IX-50 inverted microscope, 10 × magnification. B). Neutral lipid content was then quantified following spectrophotometric reading at 520 nm with SpectraMax 190 (Molecular Devices).

The effect of L-RES-nanoparticle-treated Act-MRC-5 on CRC cell growth in indirect co-culture model was tested on 3D spheroid cultures. HT-29 cells (1,500 cells/well) in 100 µL of culture medium were seeded into 96 well round bottom ultra-low attachment plates (ULA) and incubated for 3 days to form spheroids. Afterward, 100 µL of each sample of Act-MRC-5-derived CM (RES-, LIP-, and L-RES-treated Act-MRC-5 CM) was added to the tumor spheroids and incubated for a further 2 days. Bright-field images of HT-29 tumor spheroids treated with the samples were taken with an Olympus IX50 inverted microscope (Olympus, Tokyo, Japan).
To investigate the effect of L-RES-treated MCR-5* cells on the chemotherapeutic drug sensitivity of CRC cells, a combination of Act-MRC-5-derived CM (RES-, LIP-, and L-RES-treated Act-MRC-5 CM) and the chemotherapeutic drug 5-fluorouracil (5-FU) was administered in the tumor spheroid. Three-day-old HT-29 spheroids were concomitantly treated with Act-MRC-5-derived CM (RES-, LIP-, and L-RES-treated Act-MRC-5 CM) and combined with 5-FU at a concentration of 5 µM. After 48 h of incubation, the HT-29 tumor spheroid morphology was observed. The spheroid volume was quantitated using ImageJ software (Bethesda, MD, USA). The experiment was performed in triplicated wells.

The effect of SeNPs and Cs-SeNPs on 3D spheroid cultures was tested. U87 cells (1000 cells/well) in 100 µL of DMEM complete medium were seeded into 96-well round-bottom ultra-low attachment plates (cat. #7007, Corning Inc., Corning, NY, USA) and incubated for 4 days to introduce spheroids. Afterward, 100 µL of each sample (Na₂SeO₃-, SeNPs, and Cs-SeNPs) was added to the tumor spheroids and further incubated for 2 days. Bright-field images of the treated U87 tumor spheroids were taken with an Olympus IX50 inverted microscope (Olympus, Tokyo, Japan).
To investigate the effect of Cs-SeNPs on chemotherapeutic drug sensitivity, a combination of SeNPs or Cs-SeNPs and the chemotherapeutic drug 5-fluorouracil (5-FU) was administered to the tumor spheroid. The U87 spheroids were concomitantly treated with SeNPs or Cs-SeNPs at a concentration of 3.125 µg/mL and with 5-FU at a concentration of 5 µM. After 48 h of incubation, the morphology of the U87 tumor spheroid was observed, and the volume of the spheroid was quantitated using ImageJ software (NIH, Bethesda, MD, USA). The experiments were performed in triplicate.

Organoids were maintained as previously described [50 (link), 63 (link)] with modifications. Crypts from tamoxifen-treated or untreated mice were plated in 8-well chambered slides in 40μl of Matrigel at a density of ~40 crypts per Matrigel droplet. Organoids were grown for 5–7 days until fully grown. Mature organoids were passaged every 5–7 days. Images were taken using an Olympus IX-50 inverted microscope. Quantifications were performed using Image J software (National Institutes of Health).