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.

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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

  1. Dilute 33ng of PMA en 250ng of plasmid DNA in 47μL sterile H2O
  2. Add 3μL SAINT-18 and incubate for 1-2min (RT) and pipette in a 24-well
  3. Count the jurkat cells and centrifuge at max. 350xg for 10minutes
  4. Resuspend the pellet to 400,000 cells/mL in serum-free medium
  5. Add 250μL cell suspension to the well and incubate for max. 15 minutes
  6. Add minimal 500μL prewarmed complete medium
  7. Incubate at 37°C and 5% CO2 until use


Additional notes:

  1. 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.
  2. 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.
  3. The amount of SAINT-18 can be varied to increase efficiency from 2-5μL
  4. After transfection, the cells should be incubated the first 10-15 minutes in
    serum-free medium to obtain a maximum efficiency.
  5. Do not exceed this incubation period for more than 15 minutes as this leads to
    a decrease in cell viability.

 


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