After culturing cells in gels for 1 day, recombinant TNFα (100 ng/ml; Peprotech) was added. At indicated time points, cells were rapidly retrieved from alginate hydrogels by adding 30 mM EDTA in DMEM with 1% bovine serum albumin (BSA) for 5 min on ice. During this process, dead cells were labeled with the LIVE/DEAD fixable violet dead cell stain (1:1000; Thermo Fisher Scientific). Cells were then washed out with DMEM with 1% BSA and fixed with 4% paraformaldehyde in HBSS at RT for 10 min. After washing two times with PBS/0.1% BSA, cells were stained with either phospho–NF-κB p65 (Ser536) (93H1) or total NF-κB p65 (D14E12) antibody (both from Cell Signaling Technology) at 1:100 dilution in the staining buffer (HBSS/0.1% saponin/0.1% BSA) at RT for 2 hours. They were then washed out once with the staining buffer and incubated with a secondary antibody (donkey Alexa 647 anti-rabbit, Thermo Fisher Scientific) for 30 min at RT, followed by washing out and resuspension in HBSS. Flow cytometry analysis was done using LSRFortessa (BD). The sample incubated with the isotype control [rabbit immunoglobulin G (IgG), DA1E, Cell Signaling Technology] was used as a negative control. Signals from live cell fractions were used for analysis.

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