siRNA Transfection of Mouse Bone Marrow-derived Macrophages   

Abstract

Materials and Reagents

1. RPMI 1640 medium (RPMI) (Life Technologies, Invitrogen^TM, catalog number: 11875-093 )
   
2. Fetal bovine serum (Atlanta Biologicals, catalog number: S10350 )
   
3. Stock penicillin/streptomycin (P/S) (Life Technologies, Invitrogen^TM, catalog number: 15140-122 )
   
4. Lipofectamine RNAiMAX (iMAX) (Life Technologies, Invitrogen^TM, catalog number: 13778150 )
   

Equipment

1. Cell counter
   
2. 6-well plate
   
3. 24-well plate
   

Procedure

1. Isolation and culture of mouse bone marrow-derived macrophages (BMM’phi’) (Chen, 2011).
   
2. Use cell counter to count trypsinized BMM’phi’s, and then split 2 x 10⁶/ml cells into 6-well plates (for real-time PCR) or 24-well plates (for mycobacterial infection), in BMM’phi’ growth medium overnight.
   
3. Mix-1: RPMI 500 μl (6-well plate) or 100 ul (24 well plate) 40 nM siRNA.
   Mix-2: RPMI 500 μl (6-well plate) or 100 μl (24 well plate), 4.8 μl iMAx (6-well plate) or 1.2 μl iMAx (24-well plate). Add Mix-2 to Mix-1, briefly vortex and spin-down; incubate for 20 min at room temperature.
   
4. Change BMM’phi’ culture medium to RMPI 2 ml (6-well plate) or 500 μl (24- well plate), add the transfection. Mix to the well and incubate at 37 °C for 4 h.
   
5. Change to BMM’phi’ growth medium, incubate at 37 °C for 2 days. Now the cells are ready for further experiments.
   

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

1. Chen, R. (2011). Isolation and culture of mouse bone marrow-derived macrophages (BMM’phi’). Bio-protocol 1(9): e68.
   
2. Dalby, B., Cates, S., Harris, A., Ohki, E. C., Tilkins, M. L., Price, P. J. and Ciccarone, V. C. (2004). Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 33(2): 95-103.