Jurkat cells transfection protocol
Transient transfection of Jurkat T cells achieving 30% expression
Lymphocyte-derived cells are known to be difficult to transfect with non-viral vectors. Most synthetic cationic reagent of the new generation transfections reagents have proven to be unsatisfactory in the transfection of T-lymphocytes. We tried our SAINT-18 primary cell transfection reagent, which can be used for a wide variety of mammalian cells, including poor transfectable cells such as primary cells. After optimization of the transfection parameters, we developed a transient transfection protocol for activated Jurkat cells. Using this protocol we consistently achieve 30% expression of GFP in Jurkat cells using SAINT-18 primary cell transfection reagent.
Efficiency and viability were analyzed 48 hours post transfection by flow cytometric analysis (Guava Technologies). Percent eGFP positive cells after transfection with plasmid DNA (pCMV-eGFP) in combination with the stimulant PMA and the reagent SAINT-18.
Click here to read more about transfection of T-lymphocytes jurkat cells.
Transient transfection of Jurkat T cells 24-well plate format
Subculture the cells one day before transfection
- Dilute 33ng of PMA en 250ng of plasmid DNA in 47μL sterile H2O
- Add 3μL SAINT-18 and incubate for 1-2min (RT) and pipette in a 24-well
- Count the jurkat cells and centrifuge at max. 350xg for 10minutes
- Resuspend the pellet to 400,000 cells/mL in serum-free medium
- Add 250μL cell suspension to the well and incubate for max. 15 minutes
- Add minimal 500μL prewarmed complete medium
- Incubate at 37°C and 5% CO2 until use
- Prior to transfection, cells should be rigorously maintained at 0.5-1.0 million
cells/mL for 2-3 days, as density will affect both transfection efficiency as well
as cell viability.
- The amount of plasmid DNA used per transfection will vary depending on the
level of expression desired. However, every transfection should contain
approximately 200-400ng DNA.
- The amount of SAINT-18 can be varied to increase efficiency from 2-5μL
- After transfection, the cells should be incubated the first 10-15 minutes in
serum-free medium to obtain a maximum efficiency.
- Do not exceed this incubation period for more than 15 minutes as this leads to
a decrease in cell viability.