Percp5

To measure macrophage marker levels in primary TM and corticosterone-polarized BMDM flow cytometry was used. In brief, 1 × 10⁶ cells were washed twice with washing buffer (1% BSA in PBS) and incubated with an anti-mouse CD16/32 antibody (553142, BD Biosciences, San Jose, USA) for 10 min. Then, anti-mouse CD45 (103128, Biolegend, APC-R700, 1:100), F4/80 (123118, Biolegend, APC-cy7, 1:100), CD11b (553310, BD, FITC, 1:100), and CD206 (141716, Biolegend, Percp 5.5, 1:100) antibodies were used to identify the macrophage populations. After incubating the cells with CD45, CD11b, F4/80 for 30 min on ice, cells were washed with PBS 1% w/v BSA, fixed, permeabilized, and labeled with anti-CD206 at 4°C for 30 minutes. Then cells were washed and re-suspended in washing buffer. Flow cytometric analysis was performed by using a FACS AriaII (BD) and data were analyzed with FlowJo software version X (Tree Star, Ashland, OR, USA).

Monocyte subsets were analyzed by gating for CD14 (PacificBlue; BD Biosciences) and CD16 (PerCPCy5.5; BD Biosciences) in order to distinguish the three subsets of classical/inflammatory (HLA-DR⁺CD16^-CD14⁺), intermediate (HLA-DR⁺CD16⁺CD14⁺) and nonclassical (HLA-DR⁺CD16⁺⁺CD14^-) monocytes, as described earlier (27 (link)). The gating strategy is depicted in Figure S1F. Sorting of monocyte subsets was performed on a MoFlo Astrios EQ (Beckman Coulter) cell sorter. To characterize iMacs, cells were stained for CD206 (APC, eBioscience), CD45 (PE Beckman-Coulter), CD33 (PerCP5.5, Biolegend), CD14 (FITC, Beckman-Coulter), CD11b (PE BD), or with the corresponding isotype controls and analyzed on an Accuri C6 flow cytometer (BD Bioscience).

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).