Cell-Type Specific Transfection Protocols

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


Contents

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.

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