2024-10-09
ENA first public
ERC000011
ENA-CHECKLIST
BAH-GU
INSDC center name
public
INSDC status
ERS21050040
SRA accession
E-MTAB-14453:BMDM_RHplusIFNY
Submitter Id
ArrayExpress
broker name
B6 mice
cell_line
bone marrow macrophage
cell_type
not collected
collection date
house mouse
common name
Protocols: Toxoplasma gondii Tachyzoite Cell Culture  Wildtype T. gondii tachyzoites were grown at 37°C with 5% CO2 in human foreskin fibroblasts (HFFS) cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (ThermoFisher Scientific) supplemented with 3% FBS, 2mM L-glutamine and penicillin-streptomycin.    Mice C57BL/6 (B6) mice (JAX 000664) purchased from Jackson Laboratories. All mice were female, aged between 6-12 weeks.  Procedures were conducted in accredited animal facilities under the project license PPL30/3423, authorized by the UK Home Office Animal Procedures Committee.  BMDM Isolation and Infection with Toxoplasma gondii  6–12-week-old B6 mice were euthanized and femur and tibia bones were pooled from 2-3 mice. Tissue was removed from the bones using forceps, bones were placed in ethanol for 30 seconds to sterilise and then transferred to 1X sterile PBS. Bone marrow was extracted and resuspended in 1 mL ACK lysis buffer (ThermoFisher Scientific). Cells were then incubated in ACK lysis buffer for 2-5 minutes before washing. BMDMs were cultured in RPMI (ThermoFisher Scientific) complete media, supplemented with 10% filtered FBS, 2 mM of L-glutamine, penicillin-streptomycin and 50 µM ß-mercaptoethanol (ThermoFisher Scientific). Cell were diluted to 5x105 cells/mL and 30ng/mL of M-CSF (Biolegend) was added to promote BMDM differentiation. Culture media was replaced with fresh complete RPMI and 30ng/mL M-CSF 3-4 days following isolation.  After 6 days of differentiation, BMDMs were infected with wildtype RHΔKu80 T. gondii tachyzoites at an MOI of 1.5, in the presence or absence of 20 ng/mL murine recombinant interferon-γ (IFN-γ) (Biolegend). Cells were incubated at 37°C with 5% CO2 for 24 h before harvesting for scRNA sequencing.    After 6 days of differentiation, BMDMs were infected with wildtype RHΔKu80 T. gondii tachyzoites at an MOI of 1.5, in the presence or absence of 20 ng/mL murine recombinant interferon-γ (IFN-γ) (Biolegend). Cells were incubated at 37°C with 5% CO2 for 24 h before harvesting for scRNA sequencing.  Infection with Toxoplasma, both samples, than one with interferon-γ (IFN-γ), see above. Cells were washed twice with sterile PBS, then incubated with 4 mL of trypsin at 37°C with 5% CO2 for 5-7 minutes. 6 mL of complete RPMI media was then added to gently resuspended the cells. Cells were spun down at 1500 x g for 5 minutes, resuspended in 10 mL complete RPMI media and then filtered using a 40 µM filter before diluting in 0.2% Trypan Blue and counting. For 10X scRNA sequencing analysis, 1.6 x 104 cells/condition were resuspended in 0.04% BSA in PBS.  Libraries were prepared according to the 10X Genomics 3' version protocol
description
adult
developmental stage
toxoplasmosis
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
Toxoplasma gondii
infect
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
bone marrow
organism part
Mus musculus
scientific_name
IFN-y
stimulus
129 mouse strain
strain
BMDM_RHplusIFNY
title