Naïve T Lymphocyte Infection by Murine Stem Cell Virus (MSCV) Protocol   

Abstract

Materials and Reagents

1. Mouse
   
2. Fetal bovine serum (FBS)
   
3. Anti-CD3 and Anti-CD28 antibody (Ab) (Biolegend, catalog number: 100302 , 102102 ）
   
4. IL-2 (R&D systems, catalog number: 402-ML-020 )
   
5. Polybrene (Hexadimethrine bromide) (Sigma-Aldrich, catalog number: H9268 )
   
6. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
   
7. Penicillin/streptomycin solution (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
8. 2-mercaptoethanol (Life Technologies, Invitrogen^TM, catalog number: 21985-023 )
   
9. PBS solution (1x phosphate buffered saline) (Life Technologies, Gibco^®, catalog number: 10010-023 )
   
10. MSCV (Clontech, catalog number: 634401 )
    
11. RPMI-1640 medium (Life Technologies, Gibco^®, catalog number: 11875-093 ) (see Recipes)
    

Equipment

1. Centrifuges
   
2. 24-well plate
   
3. Water bath
   
4. Parafilm
   

Procedure

1. Coat plate with anti-CD3 and anti-CD28 Ab overnight.
   Add 400 μl/well PBS containing 1 μg/ml of anti-CD3 and 0.5 μg/ml of anti-CD28 to 24-well plate. Place lid or seal with parafilm, and keep in dark 4 °C overnight. Next day, discard the antibodies by aspiration and add 400 μl/well of RPMI-1640 culture medium; do not let the plate dry.
   
2. Sacrifice mouse and sort CD4+CD44 loCD62Lhi naïve T cells.
   
3. Seed 5 x 10⁶ cells/well for 24-well plate in PRMI-1640 culture medium and culture cells overnight.
   
4. Spliting cells in the ratio 1:2 (separate one well cells into two wells), adding IL-2 at final concentration of 50 U/ml, culture for 6 h (37 °C, 5% CO₂).
   
5. Spin the plate at 1,200 RPM for 5 min (room temperature), remove culture medium by aspiration, and add 1 ml RPMI-1640 with proper amount of MSCV particles (virus concentration depends on the titter which is more than 10⁶ cfu/ml) and 5-10 μg/ml of polybrene. Spin the plate at 2,500 RPM (37 °C) for 90 min.
   
6. Remove supernatant and add RPMI-1640 culture medium for differentiation or proliferation.
   

Recipes

1. RPMI-1640 culture medium
   Supplemented with
   5% FBS
   1% Penicillin/Streptomycin solution
   50 μM 2-mercaptoethanol