Cd31 pe cy7

Cells were stained with the following antibodies for analysis CD3-FITC (35–0031, Tonbo Bioscience), CD8-BV711 (100748, BioLegend), CD4-BV650 (100546, BioLegend), CD45-BUV395 (565967, BD Biosciences), CD90-BV785 (105331, BioLegend), CD11c-APC (20–0114, Tonbo Biosciences), MHC-II-Pac Blue (107620, BioLegend), CD103-PercPCy5.5 (121416, BioLegend), CD11b-A700 (557960, BD PharMingen), EpCAM-PercPe710 (46–5791-82, eBioscience), Ly51-PE (12–5891-83, eBioscience), CD31-PECy7 (25–0311-82, eBioscience), CD140a-APC (135907, BioLegend), UEA1-FITC (FL-1061, Vector Laboratories), TCRbeta-PECy7 (109222, BioLegend), CD62L-APC-Cy7 (104427, BioLegend), CD44-Alexa Fluor RTM700 (56–0441-82, BioLegend), CD25-PercP-Cy5.5 (102030, BioLegend). Annexin V staining (640906, BioLegend) was performed in Annexin V binding buffer (422201, BioLegend). Flow cytometric analysis was performed on an LSRFortessa X50 (BD Biosciences) and cells were sorted on an Aria II (BD Biosciences) using FACSDiva (BD Biosciences) or FlowJo (Treestar Software).

Tumors were dissected from the lungs of TKO Hes1^GFP/+ mice approximately 5–7 months after tumor induction and digested as previously described^{40 (link)}. The antibodies used were: CD45-PE-Cy7 (eBioscience, clone 30-F11, 1:100), CD31-PE-Cy7 (eBioscience, clone 390, 1:100), TER-119-PE-Cy7 (eBioscience, clone TER-119, 1:100), CD24-APC (eBioscience, clone M1/69, 1:200), Ncam1 (Cedarlane, clone H28-123-16, 1:100), anti-rat-IgG2a-PE (eBioscience, clone r2a-21B2, 1:200), EpCam (eBioscience, clone G8.8, 1:100), CD44-APC-Cy7 (BioLegend, clone IM7, 1:100). 1 μg/mL 7-aminoactinomycin D (Invitrogen) or DAPI was used to label dead cells.

The mouse neuroendocrine YT330 cell line used for the reprogramming experiment was sorted out from Rb/p53/p130 triple KO (TKO) Hes1^GFP/+ mice. In brief, tumours from the lungs of TKO Hes1^GFP/+ mice (6 months after tumour induction) were isolated, pooled and finely chopped with a razor blade. They were then digested in 6 ml of PBS with 120 μl of 100 mg ml⁻¹ collagenase/dispase (Roche) for 45 min with shaking at 37 °C then cooled on ice before adding 15 μl of 1 mg ml⁻¹ DNase (Sigma-Aldrich) for 5 min. The digested mixture was filtered through a 40 μm filter, pelleted and resuspended in 1 ml of red blood cell lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) for 90 s. Cells were washed once in DMEM and resuspended in FACS buffer (10% BGS in PBS, 1 million cells per 100 μl). The final single-cell suspension was stained with FACS antibodies. For negative lineage selection antibodies against CD45-PE-Cy7 (eBioscience 25–0451-82, 30-F11, 1:100), CD31-PE-Cy7 (eBioscience 25–0311-82, 390, 1:100), TER-119-PE-Cy7 (eBioscience 25–5921-82, TER-119, 1:100) were used. For positive lineage selection, antibodies against CD24-APC (eBioscience, 17–0242-82, M1/69, 1:200) were used. DAPI staining was used to identify dead cells. Cells were kept in RPMI medium for culturing.