Cells were in 3DTEBM and treated with or without Isotype/CD3, nanoBiTEs, or nanoMuTEs at a concentration of 3.7 nM for 4 days. Then, cultures were digested, and the cells were retrieved and incubated with PE anti-CD3, FITC anti-CD4, Violet anti-CD8, and APC anti-CD69 antibodies for one hour in 4°C, washed with PBS, spun down, and suspended in PBS again. These samples were analyzed by flow cytometer using MACSQuant Analyzer 10 with Ex= 488, 488, 405, and 635 nm and Em= 585/40, 525/50, 450/50, 655–730 nm, respectively. Cells were gated using FSC and SSC followed by double positive CD3+/CD4+ or CD3+/CD8+, both of which were analyzed for % of cells positive for CD69 using BD FlowJo Software.
For cytokine secretion, the supernatant was kept and the 3DTEBM was digested for two hours using collagenase following the four-day incubation period. Once the 3DTEBM was digested and samples were spun down, the supernatant (with collagenase) was then added to the supernatant collected earlier. Subsequently, the samples were analyzed for cytokine presence following the manufacturer’s protocol and scanned using the InnoScan 710 microarray fluorescence scanner (Innopsys) by the manufacturer of the cytokine array.
For cytokine secretion, the supernatant was kept and the 3DTEBM was digested for two hours using collagenase following the four-day incubation period. Once the 3DTEBM was digested and samples were spun down, the supernatant (with collagenase) was then added to the supernatant collected earlier. Subsequently, the samples were analyzed for cytokine presence following the manufacturer’s protocol and scanned using the InnoScan 710 microarray fluorescence scanner (Innopsys) by the manufacturer of the cytokine array.