Cell-Type Specific Transfection Protocols

From Heit Lab Wiki
Jump to navigation Jump to search

This page contains the protocol of transfecting specific cell types.  Unless otherwise noted, all transfections are for 12-well (22mm) plates.


RAW 264.7 Murine Macrophages

Fugene HD
  • To 100ul serum-free media add:
    • 1ug DNA, vortex gently to mix & shake/centrifuge media to bottom of tube
    • add 3ul of FugeneHD and flick tube rapidly to mix.  Contact time between fugene & the pipette tip should be minimized.
    • incubate for 15min at room temperature, then add drop-wise to the well.

Optional: Replace media in plate 4-5 hours after transfection. This will reduce cell death and cell activation.

Note: Multiple transformations of the same construct(s) can be setup in a single tube.


J774.1 Murine Macrophages

Fugene HD
  • To 100ul serum-free media add:
    • 1ug DNA, vortex gently to mix & shake/centrifuge media to bottom of tube
    • add 5ul of FugeneHD and flick tube rapidly to mix.  Contact time between fugene & the pipette tip should be minimized.
    • incubate for 15min at room temperature, then add drop-wise to the well.

Optional: Replace media in plate 4-5 hours after transfection. This will reduce cell death and cell activation.

Note: Multiple transformations of the same construct(s) can be setup in a single tube.


DC2.4 Murine Dendritic Cells

Fugene HD
  • To 100ul serum-free media add:
    • 1ug DNA, vortex gently to mix & shake/centrifuge media to bottom of tube
    • add 5ul of FugeneHD and flick tube rapidly to mix.  Contact time between fugene & the pipette tip should be minimized.
    • incubate for 15min at room temperature, then add drop-wise to the well.
    • Replace media in plate 4-5 hours after transfection. This is essential to ensure that cells survive.

Note: Multiple transformations of the same construct(s) can be setup in a single tube.


GenJet Reagent for Primary Macrophages

Manufactures Informaton.

Note: This is a non-standard use of this reagent.  Following the manufactures instructions will lead to the death of the cells.

Optional: 1 hour prior to transfection, remove media and replace with 750ul of fresh RPMI + 10% FBS

1) To 38ul of serum free media add 1.5ug of DNA

  • Multiple transfections of the same construct(s) can be setup in a single tube.
  • Mix by pipetting up and down 3-4 times.  If the dilution is high (>1:50 DNA:media [by volume]), vortex briefly to mix, then pellet mixture with a brief centrifugation

2) Into a second 1.5ml tube add 38ul of serm-free media for each well to be transfected.  Separate tubes are not needed for different combinations of DNA.

  • For each well being transformed add 2ul to 3ul of GenJet reagent.  Mix by pipetting up & down 3-4 times.
  • Immediately transfer 40ul (if 2ul of genjet used) or 41ul (if 3ul of genjet used) of this mixture into each DNA-containing tube and mix by pipetting up and down 3-4 times
    • If setting up multiple transfections in a single tube, transfer the total volume of genjet/media in a single transfer

3) Incubate for 15min at room temperature, then add drop-wise to cells

Optional: Replace media in plate 4-5 hours after transfection.  This will reduce cell death and cell activation.