Cell-Type Specific Transfection Protocols
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
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.