LLC3_PTK
Mus musculus
Protocols: MICE: Experiments were performed in 7 to 10 week-old male C57BL6/J mice (obtained from the KU Leuven animal facility or purchased from Charles Rivers). Animal housing and all experimental procedures were approved by the Institutional Animal Ethics Committee of the KU Leu-ven (Belgium) under protocol number P012/2014 and P156/2015. LLC MODEL: The syngeneic LLC cells (ATCC) were injected orthotopically into the parenchyma of the right lung through the rib cage using a 30G needle (1 × 106 cells in 70 µl matrigel (BD Biosciences)). The animals were sacrificed on day 10 of the experiment and lungs were immediately pro-cessed for TEC and NEC isolation. Mice were treated with rat anti-mouse VEGFR2 antibody DC101 (Bio-Connect) (40-50 mg/kg i.p., 3x per week), the VEGFR tyrosine kinase inhibitor PTK787 (Novartis Pharma) (100 mg/kg by daily gavage) or with, respectively, irrelevant control antibody 1C8 (Schmidt et al., 2011) (50 mg/kg i.p., 3x per week) or 5% DMSO and 1% Tween 80 in water (daily gavage) starting from day 3 after LLC injection (Liu et al., 2005; Sweeney et al., 2002). Mice were anesthetized and once the withdrawal reflex was absent in the pelvic limbs, they were perfused with 5 ml of PBS at a perfusion rate of 1 ml/min. For isolation of TECs, lung tumors were micro-dissected from lung tissue. For isolation of NECs, both lungs of control (non-tumor bearing) mice were dissected. The dissected tissue was placed into a C-tube (Miltenyi Biotec) containing 5 ml digestion buffer (0.1% collagenase II (Thermo Fisher Scientific), 0.25% collagenase type 4 (Worthington Biochemical), DNase I (150 U/min) in DMEM supplemented with 1x sodium pyruvate, 1x MEM NEAAs, ECGF/Heparin, antibiotic/antimycotic (2x) and 1% penicillin/streptomycin (Thermo Fisher Scientific)) and processed with the m_lung_01 program of gentleMACS (Miltenyi Biotec). The samples were then incubated in a water bath at 37°C for 30 min with manual shaking every 5 min. At the end of digestion, the samples were processed with the m_lung_02 program of gentleMACS, and the reaction was stopped by adding 10 ml of an isolation buffer containing 1x PBS, 0.1% BSA, 5 mM EDTA. Subsequently, the cell suspension was filtered through a 100 m cell strainer (Corning) on ice. Finally, ECs were enriched by magnetic bead sorting using MACS system (Miltenyi Biotec) at RT. Immune cells were depleted using selection with CD45 MicroBeads (Miltenyi Biotec), followed by positive selection of ECs with CD31 MicroBeads (Miltenyi Biotec), according to the manufacturer's procedures. The single cell suspensions of freshly isolated TECs and NECs (MACS-bead enriched to 20-30% as described above) were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics), aiming for 6,000 cells per library. Samples were processed using kits pertaining to V2 barcoding chemistry of 10x Genomics. Single samples were always processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel. For each experiment, all samples (TEC and NEC) were processed in parallel in the same thermal cycler.
ENA first public
2022-04-01
ENA last update
2022-04-01
External Id
SAMEA13856370
INSDC center alias
Laboratory of Angiogenesis and Vascular Metabolism VIB-KU Leuven Center for Cancer Biology Department of Oncology
INSDC center name
Laboratory of Angiogenesis and Vascular Metabolism VIB-KU Leuven Center for Cancer Biology Department of Oncology
INSDC first public
2022-04-01T12:11:00Z
INSDC last update
2022-04-01T12:11:00Z
INSDC status
public
Submitter Id
E-MTAB-7458:LLC3_PTK
age
7 to 10
broker name
ArrayExpress
cell type
endothelial cell
common name
house mouse
disease
Carcinoma, Lewis Lung
genotype
wild type genotype
organism part
lung
sample name
E-MTAB-7458:LLC3_PTK
sampling site
neoplasm
sex
male
strain
C57BL/6