Shreffler:Jurkat Transfection

Overview

We would like to use Jurkat cells as reporter cells to detect the production of retinoic acid by dendritic cells as part of the RA signaling project.

Materials

• Neisha, please edit

Procedure

Cell Preparation

1 million cells are used per transfection

1. Spin Jurkat and 3T8 cells down, resuspend in 1 mL of RPMI medium with FCS
2. Count cells ( 1 0μL of cells : 10 μL of Trypan = dilution of 2)
3. Obtain 1 million cells per cell line and incubate overnight in 10 mls of RPMI medium in 25 cm³ flask
4. Remember to keep a culture of cells for each cell type
5. Count cells the following day ( 10 μL of cells : 10 μL of Trypan = dilution of 2), and proceed with transfection

Nucleofection

1. Determine how many conditions will be done, for the last transfection 4 were done
   • Jurkat w/ RARE, Pcmv, 0.1 μg RXR/RAR
   • Jurkat w/ RARE, Pcmv, 0.4 μg RXR/RAR
   • 3T8 w/ RARE, Pcmv, 0.1 μg RXR/RAR
   • 3T8 w/ RARE, Pcmv, 0.4 μg RXR/RAR
2. This transfection was done with 4.5x10⁵ cells per condition for the Jurkat and 3T8 T-cells
3. The amount of plasmid was calculated then added into the cuvette along with the cells and the Amaxa kit solution N( 2 μg of RARE, 2 μg of Pcmv, 0.1 μg and 0.4 μg of RXR and RAR)

After the nucleofection was completed, cells were incubated overnight in a 6 well plate

Stimulation with RA

1. The following day, the cells are spun down and counted. For the last transfection, there was variation in the number of cells that survived the nucelofection:
   • Jurkat w/ RARE, Pcmv, 0.1 RXR/RAR = 3.18x10⁵ cells
   • Jurkat w/ RARE, Pcmv, 0.4 RXR/RAR = 4.95x10⁵ cells
   • 3T8 w/ RARE, Pcmv, 0.1 RXR/RAR = 2.81x10⁵ cells
   • 3T8 w/ RARE, Pcmv, 0.4 RXR/RAR = 3.31x10⁵ cells
2. For stimulation, 1x10⁵ cells were used per condition
   • 100nM RA
   • DMSO

Measuring Luciferase

1. Transfer cells to 1.5 mL eppendorfs
2. Spin cells down for 500 x g for 10 min
3. Aspirate supernatants, then added 125 μL of PLB buffer, vortex
4. Allow to shake at room temperature for 30 min
5. Spin cells at 1400 x g for 2 min, leave supernatants
6. Add 25 μL of substrate (LAR) and 5 μL of lysate (supernatants) and measure luciferase ( 14^th floor)

Discussion

discuss this protocol

References

1. Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. DOI:10.1016/0022-1759(94)90236-4 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare