Live dead fixable near ir dead cell stain kit

For the detection of CD47 expression, uninfected and infected B16F10 cells were stained with an Alexa647-conjugated antimouse CD47 antibody (Ab; clone miap301; BioLegend, San Diego, CA, USA) and the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen).
For the detection of CD86 expression and cell surface calreticulin exposure, Raw264.7 mouse macrophages were cocultured with VNP-tdT-infected or uninfected B16F10 cells for 18 h at a ratio of 1:1. At the endpoint of the incubation, the cells were harvested and stained with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen), a fluorescein isothiocyanate (FITC)-conjugated antimouse CD45 monoclonal Ab (mAb; clone 30-F11; BioLegend), a PE-Cy7-conjugated antimouse CD86 mAb (clone GL-1; BioLegend), or an anticalreticulin polyclonal Ab (pAb; ab2907; Abcam, Cambridge, UK) with an Alexa647-conjugated secondary antirabbit IgG Ab (BioLegend). To detect intracellular Salmonella, infected or uninfected B16F10 cells were fixed and permeabilized with the FOXP3 Fix/Perm Buffer Set (BioLegend) and stained with anti-S. typhimurium mAb (clone 1E6; Santa Cruz Biotechnology, Dallas, TX, USA) with an Alexa488-conjugated secondary antimouse IgG Ab (BioLegend). Flow cytometry data were acquired on a FACSCanto II flow cytometer and analyzed using FlowJo software (BD Biosciences).

Prior to antibody staining, dead cells were labelled with Live/Dead Fixable Near‐IR Dead Cell Stain Kit (Excitation 750, Emission 775; ThermoFisher Scientific) for 15 min at room temperature and excess dye washed away. Following the wash, antibodies for staining surface targets were diluted in fluorescence‐activated cell sorting (FACS) buffer and applied to cells for 30 min on ice. Unattached surface antibodies were removed with a wash. A Transcription Factor Buffer Set (BD Pharmigen) optimised for staining of intracellular targets for flow cytometric analysis was used to fix/permeabilise cells for 45 min at 4°C. Afterward, cells were washed twice in a perm/wash buffer from the same set. Antibodies for intracellular targets were diluted in perm/wash buffer, applied to cells for 45 min at 4°C, and washed twice prior to analysis on an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific). All analyses were done using FlowJo Cytometric Software (TreeStar, Inc.).