Retroviral transduction in cultured murine basophils

doi:10.1038/nprot.2008.252

Method Article

Retroviral transduction in cultured murine basophils

https://doi.org/10.1038/nprot.2008.252

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Biological techniques

basophils

retrovirus vector

cytokine

transfection

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 IMag^TM 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).

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Retroviral transduction in cultured murine basophils

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