Cell sorting set up beads for uv lasers

SupT1 cells expressing murine CTLA-4 were labelled with 5 μM carboxyfluorescein succinimidyl ester (CellTrace CFSE Cell Proliferation Kit, Life Technologies) and co-cultured overnight with human macrophages at the indicated ratios in the presence of the indicated mAbs (10 μg/mL). For blocking of FcγRs, macrophages were incubated with anti-CD32a (clone 2E08, Bioinvent) or anti-CD32b (clone 6G11, Bioinvent) F(ab’)₂ fragments at 50 μg/mL for 30 min at 37°C before adding the therapeutic antibodies and target cells. The absolute number of CFSE-labelled cells in each condition was then quantified by flow cytometry using a defined number of reference fluorescent beads (Cell Sorting Set-up Beads for UV Lasers, ThermoFisher). The percentage of killing was determined as: 100-(number CFSE⁺ targets treated/number CFSE⁺ targets untreated).

Dissected tumors were freshly cut into small pieces and resuspended in culture medium containing collagenase type 2 (1 mg/ml; Worthington Biochemical) and deoxyribonuclease I (0.1 mg/ml; Boehringer Mannheim), incubated at 37 °C with agitation for 1 h, and then filtered through a 40-mm mesh. Samples were centrifuged, washed twice with PBS, and stained for 30 min at room temperature. Antibodies used are detailed in section for reagents. For quantification of absolute number of cells, a defined number of fluorescent beads (Cell Sorting Set-up Beads for UV Lasers, ThermoFisher) was added to each sample before acquisition and used as a counting reference. Acquisition was performed with a BD LSR II Fortessa (BD Biosciences). Data analysis was conducted using FlowJo version 10.6.1 (Tree Star Inc.). For surface HER3 analysis, NTC and IRE1α-ko cells were treated with osimertinib 200 nM for 20 h and then resuspended with enzyme-free dissociation buffer (Gibco), washed 2× with cold PBS and incubated with EV20 (40 min on ice), washed 3× with cold PBS and stained with AF546-conjugated goat anti-human secondary (ThermoFisher, #A21089). Acquisition done with a BD LSR II Fortessa (BD Biosciences). Data analysis using FlowJo version 10.6.1 (Tree Star Inc.).