Lsrfortessa x 20 instrument

MDA-MB-231 and MCF7 cells were treated with vehicle or 20 μM or 40 μM imipramine for 96 h and then incubated with 10 μM BrdU for 2 h to pulse-label the cells. Cells were harvested and then fixed with cold ethanol. Cells were then washed with phosphate-buffered saline (PBS) and incubated with 2 N HCl/0.5% Triton X-100 for denaturation before neutralization with 0.1 M sodium borate (Na₂B4O₇, pH 8.5). Samples were incubated with anti-BrdU antibody (BD, Cat #347580) for 60 min at room temperature. Samples were washed and then incubated with Alexa647-labeled goat anti-mouse secondary antibody (Thermo Fisher, Cat # A-21236) for an additional hour. Samples were washed again and resuspended in PBS containing propidium iodide to label DNA before performing flow cytometry using a BD LSRFortessa^™ X-20 instrument. Data were processed and the percentage of BrdU-positive cells calculated using FlowJo v10.7.2 software.

B16F10 cells expressing full-length human MICA or MICB were collected, counted and plated at a density of 2 × 10⁵ cells per well in a 96-well V-bottom plate. All centrifugation steps were performed at 300g for 5 min. Cells were washed twice with PBS, pelleted and stained with 100 μl Zombie Green dead cell exclusion dye (diluted 1:800 in PBS; BioLegend) for 20 min at room temperature. Cells were washed with PBS containing 2% FBS and 2 mM EDTA (FACS buffer) and incubated with 100 μl of serum samples serially diluted in FACS buffer for 1 h at room temperature. Following two washes, cells were stained with 100 μl of Alexa Fluor 647-conjugated goat anti-mouse IgG (1 μg ml⁻¹ in FACS buffer; BioLegend) for 30 min at room temperature. Samples were washed twice with FACS buffer. Samples were analysed using an LSRFortessa X-20 instrument (BD Biosciences) and FlowJo software. Sera from control immunized mice, parental cells without MICA or MICB expression and respective secondary antibodies were used as negative controls for flow cytometry.
A similar staining procedure was followed for assays using macaque serum samples with the following modifications. HEK293T cells expressing rhesus macaque MICA or MICB protein were stained with serum samples, and 100 μl of diluted (1 μg ml⁻¹ in FACS buffer) PE-conjugated goat anti-rhesus macaque IgG (Southern Biotech) was used for detection.