Retroviral Infection of T cells
Van Parijs Lab
January 2001
Day 1
1. Culture 293T cells at 1.5 – 2.0 X106 cells/ 4 mL of DIO in a 60mm petri dish
Day 2 Calcium phosphate transfection of 293T cells
(cells should be approximately 80 percent confluent)
1. In a 5mL sterile tube, combine 400 l ddH2O, 100 l 1.25 M CaCl2
2. Add DNA
Retroviral DNA 7-10g
PCLEco
3-5 g
*maximum of 12g total
3. Add 500l 2X HBS (pH 7.05) dropwise. Bubble continually while adding and for 15
seconds after adding.
4. Immediately transfer HBS and DNA onto cells, dropwise. Swirl to cover the plate
uniformly.
5. After 4-20 hours, replace media with 4 mL prewarmed D10
*transfection efficiency should be 50-70 % for optimal infection. If lower, increase
time of transfection and/or try a different batch of HBS. If cells look poor after
transfection, decrease time of transfection.
Day 3 Activate T cells
1. Harvest spleen and/or lymph nodes from mouse. Place into a 60 mm petri dish with
4mL CIO. Use screens and syringes to make a single cell suspension.
2. Resuspend in TAC (5 mL/spleen, 3 mL/LN from one mouse)
3. Incubate 5 minutes at room temperature.
4. Spin and wash 1X with CI0, Count cells (deplete CD8+ or CD4+ T cell population if
required)
5. Culture 2 X 106 cells/mL CIO with 1g/mL CD3 or 1g/mL peptide (for TCR
transgenic cells), in a 24-well plate.
Day 4 Infection #1
1. Collect supernatant from 293 cells into sterile tube and replace with pre-warmed
DIO.
2. Add to supernatant 10 l/ml Hepes (100X) and 10l/mL of polybrene
(1mg/mL=100X)
3. Spin retroviral supernatant 2 minutes at 2000 RPM to remove detached 293T cells
4. Remove 70 % of supernatant from T cell culture wells and add 1mL retroviral
supernatant/well
5. Wrap 24-well plate in saran wrap to prevent contamination. Spin cells for 1 hour at
2500 RPM at 30 degrees C.
6. Remove 70-80% of retroviral supernatant. Add 1mL of C10 containing 1g/mL
CD3 OR 1g/mL peptide AND 1ng/mL IL-2
Day 5 Infection #2

Repeat Infection (if required)
Day 6

Analyze T cells
D10
440 mL DMEM
50 mL FBS
5 mL L-glutamine (100X)
5 mL Pen/Strep (100X)
-Sterile Filter
2X HBS
50mM Hepes pH 7.05
10mM KCl
12mM Dextrose
280mM NaCl
1.5mM Na2HPO4
-pH to 7.05+0.05 (it is critical that the pH be accurate)
-Sterile Filter
CIO
420 mL RPMI
50 mL FBS
5 mL L-glutamine (100X)
5 mL Pen/Strep (100X)
5 mL NEAA (100X)
5 mL Sodium Pyruvate (100X)
5 mL Hepes (100X)
5 mL 2-ME (100X = 5.5X10-6M)
-Sterile Filter
TAC
450 mL .16 M NH4Cl
50 mL .17 M Tris (pH 7.4)
-Sterile Filter