Cytofix cytoperm fixation permeabilization buffer

After 48 h of treatment, the cell co-cultures were incubated in Accutase (at 37 °C for 15 min) to dissociate huTGOs into single cells as previously published [30 (link)]. All cells were collected by centrifuging at 300× g for 5 min, then resuspended in 100 µL of diluted Zombie UV dye (1:100 in PBS) and stained for 15 min at RT. The cells were incubated at 4 °C for 30 min with fluorochrome-conjugated antibodies specific for CD8a, CD14, CD15, CD11b, CD33, CD137, EpCAM, granzyme B, Perforin, HLA-DR, and PD-L1 (1:100 dilution, all from BioLegend), diluted in 100 μL cell staining buffer. Cells were washed with cell staining buffer (BioLegend) and incubated with the Cytofix/Cytoperm Fixation/Permeabilization Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 °C. Cells were then washed and resuspended in 100 µL of cell staining buffer and stained at 4 °C for 30 min with fluorochrome-conjugated intracellular antibodies specific for perforin, IL-2 and interferon-gamma (IFN-g) (both from BioLegend) diluted in 100 µL cell staining buffer. Cells were washed, resuspended in 300 µL of cell staining buffer, filtered, and then analyzed on an LSRII system (BD Biosciences). An unstained cell sample and single stained beads for each antibody were used as gating controls. Data were analyzed using FlowJo software (BD Biosciences).

For intracellular cytokine staining, stimulated cells were stained first with fluorochome-labeled monoclonal antibodies to surface markers. After being permeabilized with cytofix/cytoperm fixation/permeabilization buffer (BD Biosciences), the cells were incubated with fluorochrome-labeled monoclonal antibody to human IFN-γ, IL-17F, TNF-α, or granzyme B for 30 minutes at 4 °C. Appropriate isotype-matched control antibodies were used to determine background levels of staining. The expression of cytokines and granzyme B was analyzed by an FC-500 flow cytometer (Beckman Coulter).

Cells were fixed and permeabilized with BD Cytofix/Cytoperm fixation/permeabilization buffer according to the manufacturer’s instructions. Envelope antigen was then detected with a 1:400 dilution of Alexa fluor 488-conjugated mouse monoclonal antibody 4G2 (Novus Biologicals) for 25 min at 4 °C. Cell fluorescence was then analyzed on a Macsquant VYB Flow Cytometer (Miltenyi Biotec), by using FCS and FITC fluorescence parameters, and by subtracting cell autofluorescence background. Data were analyzed with FlowJo (BD) software.