Evos floid imaging system

HCT116 colon cancer cells were seeded in 24-well plates with a density of 1 × 10⁵ cells and incubated overnight before the treatment. The cells were treated with the IC50 concentration of dolasetron and ketoprofen and incubated for 48 h. After washing the control and drug-treated wells with PBS, the cells were stained with a mixture of acridine orange and ethidium bromide (100 µg/mL each). The stained cells were visualized and imaged immediately using an inverted fluorescent microscope, and images were captured using the Thermo Fisher scientific EVOS Floid imaging system [25 (link)]. For apoptosis, caspase-3/7activity was measured using the Apo-one Homogeneous Caspase-3/7 assay kit (Promega Madison, WI, USA; cat no. G7790), and the manufacturer’s protocol was followed. Cleavage of the non-fluorescent substrate, Z-DEVD-Rhodamine-110, by caspase 3/7 resulted in fluorescent rhodamine-110. The fluorescence of the sample was measured at 530 nm emission and 490 nm excitation in the multimode reader, BioTek [26 (link),27 (link)].

In a 96 well plate (cat# 167008, Thermo Fisher Scientific), 100 μL of media containing 1x10⁴ primary PBMCs were added to 100 μL of media with or without CD27xEGFR (10 µg/mL) containing MDA-MB-231^WT or MDA-MB-231^scFvCD3 cells previously incubated overnight at Effector : Target (E:T) ratios of 1:1, 1:2 and 1:5. After a 24-hour incubation, images were taken at 5x magnification using an EVOS FLoid Imaging System (cat# 4471136, Thermo Fisher Scientific) to visualize T cell clustering. Furthermore, PBMC cells were collected, stained for CD3 and CD25 and the CD25 expression of CD3⁺ cells was measured using flow cytometry (CytoFLEX V5-B5-R3).

HCT116 cells were plated on a coverslip held in a 6-well tissue culture plate, and the cells were allowed to grow overnight. Subsequently, the cells were treated with the IC50 concentration of dolasetron and ketoprofen and incubated for 24 h. Following treatment, the cells were fixed with 10% formalin and permeabilized with 1% Triton X-100 in PBS. Later, the cells were incubated with 5% bovine serum albumin in PBS for 30 min. To detect specific proteins, the cells were incubated with a 1:50 dilution of PUM1 antibody (Abcam, Cambridge, UK; ab92545), DCLK1 antibody (Abcam, Cambridge, UK; cat no. ab37994), and CD133 antibody (Cell Signaling Technology Danvers, Beverly, MA, USA; cat no. 5860s). The primary antibody was detected using a Goat anti-Rabbit IgG (H + L) Cross-Absorbed Secondary Antibody, Alexa Fluor 633 (Thermo Fisher Scientific, Eugene, OR, USA; A21070). Fluorescence images were captured using the EVOS FLoid Imaging System, from Thermo Fisher Scientific [28 (link)].

TPC and BCPAP cells underwent immunocytochemistry using Anti-ZEB1 (NBP1-05987) following the manufacturer’s instructions. Briefly, cells were washed with PBS following DMEM removal, fixed with 4% PFA for 20 minutes at room temperature, and then washed thrice with PBS. A blocking solution (300 mg BSA, 30 μL Triton X-100, 10 mL PBS 1x) was used for incubation for 1 hour. Primary antibody was applied to coverslips and incubated overnight at 4 °C, followed by three PBS washes. Subsequently, a secondary antibody was added and incubated for 1 hour. Finally, DAPI (300 nM) staining was performed with a 20-minute incubation. Images were acquired using the EVOS FLoid Imaging System (ThermoFisher Scientific).

6–16 h post-transfection of the construct expressing EGFP, the fluorescence of each well was verified and imaged using EVOS FLoid Imaging System (Thermo Fisher Scientific). 48 h post-transfection, the medium of each well was gently aspirated, followed by the addition of 100 µl/well TrypLE Express (Thermo Fisher Scientific) and incubated at 37 °C for 10 min, then diluted with 100 µl/well culture medium (1 % (v/v) FBS). Flow cytometry was performed using FACSCanto II Flow Cytometer (BD Biosciences) and analyzed using FlowJo 10.7.1 (FlowJo, LLC). Cells were gated by forward versus side scatter (FSC vs. SSC) plot to identify cell population and exclude debris, forward scatter height versus forward scatter area (FSC-H vs. FSC-A) plot for doublet exclusion, and FSC-H or histogram vs. FITC-A plot to reflect EGFP signal. All represented samples were assayed with three biological replicates. Data is representative of at least 5,000 gated events per condition.

Immunofluorescence was performed using the lung cancer A549 cell line as the positive control because they express immune checkpoint receptors on their surface and the HEK293 cell line as the negative control because it is not a tumor cell. In total, 1 × 10⁵ cells were plated in sterile 24-well plates on circular glass slides to allow cell adhesion and spreading until reaching confluency. Recombinant TIM-3-ECD (50 μg/mL) was then added to the culture medium and incubated at 37 °C for 1 h. After incubation, cells were washed twice with 1 mL of serum-free culture medium and incubated with 1 mL of chilled 4% paraformaldehyde at 4 °C for 48 h for fixation. Next, cells were washed twice with 1 mL of PBS 2% glycine buffer and blocked with 1 mL of PB1% at 4 °C for 18 h. After three washes with 1 mL of PBS, FITC-conjugated His-Tag-specific antibody diluted to 1:200 in PB1% was added and incubated at room temperature for 1 h under protection from direct light. Glass slides were washed with 1 mL of PBS three times and then removed from the plate and sealed with SlowFade Diamond reagent (Thermo Fisher Scientific, United States). The reactions were visualized on a fluorescence microscope with a green filter (emission and excitation wavelengths of 482 nm and 532 nm, respectively) using an EVOS FLoid Imaging System (Thermo Fisher Scientific, United States) at ×10 magnification.