Return to study ST001986 main page

MB Sample ID: SA185847

Local Sample ID:RH2-06
Subject ID:SU002067
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:Fbxo7LacZ mice (Fbxo7tm1a(EUCOMM)Hmgu on a C57BL/6J background) were bred as heterozygous crosses
Age Or Age Range:WT, heterozygous and homozygous littermates were harvested between 6-9 weeks.
Gender:Male and female
Subject Comments:All experiments in mice were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and ARRIVE guidelines. Animal licences were approved by the Home Office and the University of Cambridge's Animal Welfare & Ethical Review Body Standing Committee. Experiments were performed under Home Office licence PPL 70/9001.

Select appropriate tab below to view additional metadata details:

Collection:

Collection ID:CO002060
Collection Summary:Spleens were harvested from WT, heterozygous or homozygous Fbxo7LacZ mice and processed to a single cell suspension. For n=2 of Fig. 3E, cells were incubated in RBC lysis buffer (eBioscience) for 5 minutes prior to centrifugation. For all other data, viable lymphocytes were separated with mouse Lympholyte® Cell Separation Media (Cedarlane Labs). CD4+ T cells were then isolated by negative selection using the MojoSort Mouse CD4 T Cell Isolation Kit (Biolegend). Isolated CD4+ T cells were seeded at 1x106/mL in RPMI supplemented with 10% FBS, 100 U/mL penicillin and streptomycin and 5 µM β-mercaptoethanol. To activate, cells were added to plates coated with 2 µg/mL α-CD3 (clone 145-2C11) and containing 2 µg/mL soluble α-CD28 (clone 37.51) and incubated for the indicated duration. Cell viability and activation were measured by flow cytometry by staining with antibodies to CD4-PE (clone GK1.5), CD25-PE/Cy7 (clone PC61), CD69-FITC (clone H1.2F3) and an eFluor™ 780 fixable viability dye (eBioscience) for 30 min at 4oC in the dark. Samples were analysed on a CytoFLEX S flow cytometer.
Sample Type:T-cells
  logo