Eight week-old male B6.A2G-Mx1 and B6.A2G-Mx1-Tyk2−/− were used to generate Hoxb8 neutrophil cultures as described (49 (link)). Briefly, myeloid progenitor cells were derived from bone marrow, retrovirally transduced with an estrogen-regulated Hoxb8 construct (MSCV-ERHBD-Hoxb8 (50 (link)) and selected for 4 weeks in the presence of stem cell factor (SCF) to generate neutrophil progenitor lines. Polyclonal progenitor cell lines were cultured in OptiMEM + GlutaMAX medium (Life Technologies) supplemented with 10 % FCS, 30 μM β-mercaptoethanol (Life Technologies), 1 μM β-estradiol (Sigma-Aldrich) and 1 % supernatant from SCF-producing CHO cells. Differentiation was induced by β-estradiol removal in the presence of 1% SCF supernatant and 20 ng/ml murine recombinant G-CSF (Peprotech). After 3.5-4 days of differentiation cells were used for experiments. For IFN treatment, cells were treated for 4 h with the indicated concentrations of either IFN-αB/D or recombinant mouse IFN-λ2 and then processed for RNA isolation and subsequent RT-qPCR analysis.
Protocol detail
Hoxb8-Induced Neutrophil Differentiation and Interferon Response
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Bone marrow
Cell lines
Cells
Cells differentiation
Cultured medium
Estradiol
Estrogen
G csf
Isolation
Male
Mercaptoethanol
Mouse
Myeloid progenitor cells
Neutrophil
Progenitor cell
Stem cell factor
Tyk2
Corresponding Organization : University of Freiburg
Other organizations : The Francis Crick Institute, Heidelberg University, Institute of Medical Microbiology and Hygiene, Aarhus University, University of Veterinary Medicine Vienna
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Variable analysis
independent variables
- Mouse genotype: B6.A2G-Mx1 and B6.A2G-Mx1-Tyk2−/−
- IFN treatment: IFN-αB/D or recombinant mouse IFN-λ2
- IFN treatment concentration: Indicated concentrations
- Gene expression: Measured by RT-qPCR analysis
- Cell type: Hoxb8 neutrophil progenitor cells
- Cell culture conditions: OptiMEM + GlutaMAX medium, 10% FCS, 30 μM β-mercaptoethanol, 1 μM β-estradiol, 1% SCF supernatant
- Differentiation conditions: β-estradiol removal, 1% SCF supernatant, 20 ng/ml G-CSF
- IFN treatment duration: 4 hours
- Positive control: Not specified
- Negative control: Not specified
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