Authors: Shigeaki Hida & Shinsuke Taki

### Introduction 

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.

### Reagents 

1. 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)

### Equipment

1. Flowcytometer (FC500, Beckman-Coulter)
- BD IMagTM Cell Separation System (BD Bioscience)
- AutoMACS (Myltenyi Biotech)

### Procedure

**Preparation of retrovirus-containing supernatant**

- 1. 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.
- 2. Collect retrovirus-containing supernatants 48 hours after transfection, and concentrate 10-fold by centrifugation (8000 g, 12 hours, 4°C).
- 3. 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**

- 4. Isolate bone marrow (BM) cells from femurs and tibias of 8-12 week old mice.
- 5. 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.
- 6. Enrich BM-derived basophils by depleting mast (c-kit+) cells using IMag system or autoMACS as instructed by the supplier.
- 7. 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.
- 8. 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.
- 9. Recover infected cells and wash with RPMI 1640 containing 2% FBS.
- 10. 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.

### Anticipated Results 

See Figure 1.

### References

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).

### Figures
 
**Figure 1: Retroviral reconstitution of FcRγ allows surface expression of FcεRI on BM-derived basophils**. 

![Fig 1](http://i.imgur.com/YjXbtQ1.png "Fig 1")

*BM-derived basophils from FcRγ-deficient mice were infected with either control or FcRγ-expressing retrovirus. Infected cell, deleted of c-kit+ cells, were stained with anti-FcεRIα (MAR-1, e-Bioscience) and anti-rat CD2 (OX34, Cedarlane) and analyzed on the FC500 flowcytometer*.

### 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**, Shigeaki Hida, Sho Yamasaki, Yuzuru Sakamoto, Masaya Takamoto, Kazushige Obata, Toshiyuki Takai, Hajime Karasuyama, Kazuo Sugane, Takashi Saito, and Shinsuke Taki, *Nature Immunology* 10 (2) 214 - 222  21/12/2008  [doi:10.1038/ni.1686](http://dx.doi.org/10.1038/ni.1686)

### Author information
 
**Shigeaki Hida & Shinsuke Taki**, Department of Immunology and Infectious Diseases, Shinshu University Graduate School of Medicine, Japan 

*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/510) (2008) doi:10.1038/nprot.2008.252. Originally published online 30 December 2008*.