This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Retroviral transduction in cultured murine basophils
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
Basophils are one of effector cells in type 2 immune responses that critically are associated with allergic inflammation and infections with helminth parasites. Recent findings that basophils produce a large amount of Th2 cytokines (IL-4 and IL-6) has provided new insights into the possible role of basophils in the initiation phase, in addition to the effector phase, of type 2 immune responses. However, basophils occupy only 1% or less in blood, bone marrow and spleen, making it difficult to analyze their function.
Here we establish a method for expressing exogenous proteins and their mutants in cultured basophils through retroviral infection. In combination with cultured basophils from gene-manipulated mice, this method, overcoming the paucity of basophils, allows us to examine the requirement of various signal transducers in interleukin 3-induced production of Th2 cytokines.
– RMPI 1640
– Fetal bovine serum (FBS)
– Recombinant mouse interleukin-3
– Biotin-conjugated anti-c-kit antibody (e-Bioscience)
– Biotin-conjugated anti-rat CD2 (rCD2) antibody (Cedarlane)
– PE-Cy7-streptavidin (BD Biosciences)
– RetroNectin (TakaraBio)
– FuGene-6 reagent (Roche Diagnostics)
– pMX-IRES-rCD2 (The GFP portion in the original pMX-IRES-GFP vector was replaced with the extracellular-transmembrane domain of rCD2; see ref. 1)
– Flowcytometer (FC500, Beckman-Coulter)
– BD IMagTM Cell Separation System (BD Bioscience)
– AutoMACS (Myltenyi Biotech)
Preparation of retrovirus-containing supernatant
- Transfect retroviral constructs (based on the bicistronic vector, pMX-IRES-rCD2, see ref. 1) into the packaging cell line Phoenix using FuGene-6 reagent as instructed by supplier.
- Collect retrovirus-containing supernatants 48 hours after transfection, and concentrate 10-fold by centrifugation (8000 g, 12 hours, 4°C).
Filtrate virus supernatants through 0.22μm pore PVDF membrane. The filtrated supernatants can be stored for up to one week at 4°C.
Infection of BM-derived basophils with retroviruses
- Isolate bone marrow (BM) cells from femurs and tibias of 8-12 week old mice.
- Culture whole BM cells at 2 to 2.5 × 106 cells/ml in 10 ml of penicillin and streptomycin, 2mM L-glutamine, 50 μM β-mercaptoethanol, 10% fetal bovine serum-containing RPMI 1640 medium supplemented with recombinant mouse IL-3 (5 ng/ml) for 9 days with medium changed every 3 days.
- Enrich BM-derived basophils by depleting mast (c-kit+) cells using IMag system or autoMACS as instructed by the supplier.
- Treat 12-well plates with RetroNectin solution (50 µg/ml in PBS) for 2 hours at room temperature, followed by 2% bovine serum albumin in PBS for 30 minutes.
- Incubate the treated plates with the virus supernatants for 4 hours at 30°C. Add enriched BM-derived basophils (0.5 to 1 × 106 cells/ml) and culture for 2 days for infection.
- Recover infected cells and wash with RPMI 1640 containing 2% FBS.
- For stimulating with IL-3, infected BM-derived basophils are ‘starved’ by culturing in culture media without IL-3 for 12 to 18 hours before enrich rat CD2+ cells with IMag system and/or AutoMACS.
See Figure 1.
- Yamasaki, S. et al. Mechanistic basis of pre-T cell receptor-mediated autonomous signaling critical for thymocyte development. Nat. Immunol. 7, 67-85 (2006).
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Dec, 2008
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
Associated Publications
Fc receptor gamma-chain, a constitutive component of the IL-3 receptor, is required for IL-3-induced IL-4 production in basophils
by Shigeaki Hida, Sho Yamasaki, Yuzuru Sakamoto, +6
Nature Immunology (03 November, 2008)
Method Article
Retroviral transduction in cultured murine basophils
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Dec, 2008
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Figure 1
Basophils are one of effector cells in type 2 immune responses that critically are associated with allergic inflammation and infections with helminth parasites. Recent findings that basophils produce a large amount of Th2 cytokines (IL-4 and IL-6) has provided new insights into the possible role of basophils in the initiation phase, in addition to the effector phase, of type 2 immune responses. However, basophils occupy only 1% or less in blood, bone marrow and spleen, making it difficult to analyze their function.
Here we establish a method for expressing exogenous proteins and their mutants in cultured basophils through retroviral infection. In combination with cultured basophils from gene-manipulated mice, this method, overcoming the paucity of basophils, allows us to examine the requirement of various signal transducers in interleukin 3-induced production of Th2 cytokines.
– RMPI 1640
– Fetal bovine serum (FBS)
– Recombinant mouse interleukin-3
– Biotin-conjugated anti-c-kit antibody (e-Bioscience)
– Biotin-conjugated anti-rat CD2 (rCD2) antibody (Cedarlane)
– PE-Cy7-streptavidin (BD Biosciences)
– RetroNectin (TakaraBio)
– FuGene-6 reagent (Roche Diagnostics)
– pMX-IRES-rCD2 (The GFP portion in the original pMX-IRES-GFP vector was replaced with the extracellular-transmembrane domain of rCD2; see ref. 1)
– Flowcytometer (FC500, Beckman-Coulter)
– BD IMagTM Cell Separation System (BD Bioscience)
– AutoMACS (Myltenyi Biotech)
Preparation of retrovirus-containing supernatant
- Transfect retroviral constructs (based on the bicistronic vector, pMX-IRES-rCD2, see ref. 1) into the packaging cell line Phoenix using FuGene-6 reagent as instructed by supplier.
- Collect retrovirus-containing supernatants 48 hours after transfection, and concentrate 10-fold by centrifugation (8000 g, 12 hours, 4°C).
Filtrate virus supernatants through 0.22μm pore PVDF membrane. The filtrated supernatants can be stored for up to one week at 4°C.
Infection of BM-derived basophils with retroviruses
- Isolate bone marrow (BM) cells from femurs and tibias of 8-12 week old mice.
- Culture whole BM cells at 2 to 2.5 × 106 cells/ml in 10 ml of penicillin and streptomycin, 2mM L-glutamine, 50 μM β-mercaptoethanol, 10% fetal bovine serum-containing RPMI 1640 medium supplemented with recombinant mouse IL-3 (5 ng/ml) for 9 days with medium changed every 3 days.
- Enrich BM-derived basophils by depleting mast (c-kit+) cells using IMag system or autoMACS as instructed by the supplier.
- Treat 12-well plates with RetroNectin solution (50 µg/ml in PBS) for 2 hours at room temperature, followed by 2% bovine serum albumin in PBS for 30 minutes.
- Incubate the treated plates with the virus supernatants for 4 hours at 30°C. Add enriched BM-derived basophils (0.5 to 1 × 106 cells/ml) and culture for 2 days for infection.
- Recover infected cells and wash with RPMI 1640 containing 2% FBS.
- For stimulating with IL-3, infected BM-derived basophils are ‘starved’ by culturing in culture media without IL-3 for 12 to 18 hours before enrich rat CD2+ cells with IMag system and/or AutoMACS.
See Figure 1.
- Yamasaki, S. et al. Mechanistic basis of pre-T cell receptor-mediated autonomous signaling critical for thymocyte development. Nat. Immunol. 7, 67-85 (2006).
Associated Publications
Fc receptor gamma-chain, a constitutive component of the IL-3 receptor, is required for IL-3-induced IL-4 production in basophils
by Shigeaki Hida, Sho Yamasaki, Yuzuru Sakamoto, +6
Nature Immunology (03 November, 2008)