2024-10-01
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA115120927
External Id
Institute of Immunobiology, Kantonsspital St. Gallen, St.Gallen, Switzerland
INSDC center name
2024-01-20T10:54:12Z
INSDC last update
public
INSDC status
ERS17784332
SRA accession
E-MTAB-13708:Sample 14
Submitter Id
6 to 10
week
age
ArrayExpress
broker name
cytotoxic T cell
cell_type
not collected
collection date
house mouse
common name
Protocols: Mice were sacrificed at age 6-10 weeks and tissue from lung or excised tumors on day 23 were disassembled and digested for further stromal cell or immune cell isolation and scRNA sequencing analysis. Tumor cell growth protocol: a murine lung cancer cell line Lewis lung carcinoma (LLC) line expressing H-2Db–restricted peptide 33-41 of the lymphocytic choriomeningitis virus (LCMV) glycoprotein (LLC-gp33) was used. LLC-gp33 cells were cultured in Iscove modified Dulbecco medium containing antibiotics and selection agents (Sigma-Aldrich). LLC-gp33 cells together with the fluorescent reporter protein tdTOMATO (tdTOM) were intravenously injected into Ccl19-EYFP mice. After 23 days lungs were harvested, subpleural tumor nodules were counted under the stereomicroscope and lungs were weighed. The mean weight of tumor-free lungs was subtracted for calculation of tumor weight per lung. Ccl19-EYFP+ cells from naïve lungs and established tumors on day 23 were isolated and further processed for single cell RNA-sequencing. Murine recombinant coronavirus vector (mCOV) treatment protocol: The recombinant murine coronavirus vector (mCOV) expressing the LCMV glycoprotein gp33 and the myeloid cell stimulating factor Flt3l (mCOV-Flt3l-gp33)(Perez-Shibayama et al, 2013) was used to boost anti-tumor T cell response. Mice were anaesthesized with isofluran and 5 x 104 plaque forming units (pfu) (in medium) were applied to the nose for inhalation. Diphtheria toxin mediated ablation of FRC progenitors protocol: Ccl19-EYFP/DTR mice were injected with LLC-gp33-TOMATO cells and were anaesthesized with isofluran. Intranasal inhalation of 10 ng of diphtheria toxin on days 10 and 12 after tumor cell injection in lungs of Ccl19-iEYFP/iDTR mice, followed by mCOV-Flt3l-gp33 immunization on day 15 was used for cell depletion. For enzymatic digestion of disaggregated lung tissue from tumors or excised tumors (day 23), 1 mg/ml Dispase (Roche), 1 mg/ml Collagenase Type II (Gibco) and 25 μg/ml DNaseI (AppliChem) in combination with gentleMACS-based mechanical disruption (Miltenyi Biotech) were used. After 30 minutes incubation at 37°C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Enrichment of stromal cells was achieved by depleting hematopoietic and erythroid cells using MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec). The flow-through was centrifuged and single cell suspension was stained for further flow cytometric analysis or cell sorting. The unbound single cell suspension was stained for cell sorting with a BD FACS Melody 2-way or 4-way cell sorter (BD Biosciences) and run on the 10x Chromium analyzer (10X Genomics). For the isolation of immune cells, tissues were gently smashed through a mesh using a syringe plunger and washed with PBS containing 0.5% FCS and 10 mmol/L EDTA before staining for flow cytometric analysis or cell sorting. cDNA library generation was performed following the established commercial protocol for Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3/3.1 Chemistry.
description
adult
developmental stage
Tg(Ccl19-Cre)+ x R26-EYFP++
genotype
not collected
geographic location (country and/or sea)
mixed pool of 4 to 10 individuals
individual
not applicable
isolate
Yes
mcov treatment
Mus musculus
organism
Mus musculus
organism
lung tumor
organism part
Mus musculus
scientific_name
mixed
sex
C57Bl/6N
strain
Sample 14
title