Phagocytosis Assay for Dendritic Cell-Mediated Killing of NSCLC Cells

After in vitro generation of DCs, NCI-H1975, A549 and NCI-H1650 cells were labelled with PKH67, a green fluorescent membrane dye (Sigma Aldrich, MIDI67), prior to plating them out on day 3. NSCLC cells were treated with chemotherapy on day 4. On day 5, immature DCs were stained with cytoplasmic violet-fluorescent CellTracker Violet BMQC dye (Invitrogen, C10094) and effector (E) and target (T) cells were placed in coculture at a 1:1 (E:T) ratio. Supernatant (SN) was stored (−20 °C), cells were collected and immediately used for flowcytometric detection of DC maturation markers and phagocytosis on day 7. Cells were stained with CD80-PerCP5.5 (Biolegend, 400150) and CD86-PE-Cy7 (BD Biosciences, 557872) to assess DC maturation (Violet⁺ population). Isotype controls (PerCP5.5, Biolegend, 305232; PE-Cy7, BD Biosciences, 557872) were included to subtract aspecific signals from measured fluorescence intensity. Phagocytosis of NSCLC cells was assessed by gating on the PKH67⁺Violet⁺ population, as previously described [35 (link)]. Acquisition was performed on a FACSAria II (BD Biosciences). Data analysis was performed using FlowJo v10.1 software (TreeStar).