Lightning link rapid antibody labeling kit

To allow LC3-II accumulation, PBMCs were treated with lysosome-targeting Bafilomycin A1 (InvivoGen) at 50 nM for 4 h or left untreated^{36 (link)}. To determine autophagy flux upon TCR-mediated activation, PBMCs were first activated for 16 h with soluble 2 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (both Sanquin) followed by incubation with 50 nM Bafilomycin A1 for 4 h. Anti-LC3 monoclonal antibody was conjugated to AF488 using the Lightning-Link® Rapid Antibody Labeling kit (Expedeon) according to the manufacturer’s instructions. Viable cells were suspended in 0.05% saponin (Sigma-Aldrich) for 15 min at room temperature to flush out LC3-I^{36 (link)}. Cells were washed twice with PBS, then stained with the conjugated LC3-AF488 in combination with antibodies against surface receptors in 0.05% saponine buffer for 30 min, shielded from light, shaking at 600 strokes/min at 4 °C. After incubation, cells were washed twice with PBS, then fixated using 4% PFA, washed, stored in PBS and acquired on a Fortessa Cell Analyzer.