Alexa fluor 647 conjugated goat anti mouse igg

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.

Flow Cytometry Standard (FCS) files of CD4 T cells fixed, permeabilized and stained with anti-granzyme A mAb (BioLegend) or IgG control, Alexa Fluor 647-conjugated goat anti-mouse IgG, blocked with mouse IgG, stained with Alexa Fluor 488-conjugated anti-CCR7 (BioLegend) or IgG control were acquired using an LSRII flow cytometer (Becton Dickenson) with FACSDiva software (Becton Dickenson) and analyzed using Flowjo software (Tree Star).