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.

1 million cells are used per transfection

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

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
This transfection was done with 4.5x10⁵ cells per condition for the Jurkat and 3T8 T-cells
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

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
For stimulation, 1x10⁵ cells were used per condition
- 100nM RA
- DMSO

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