J774 Cell Transfection

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Contents

Protocol

Cell Culture
  1. Prepare a 6-well plate by adding 2 ml of J774 Cell media per well. If coverslips are required, add one coverslip/well prior to adding the media.
  2. Wash the cells pre-cultured in a T25 flask with 5 ml of PBS and trypsinize with 1.5 ml of trypsin for five minutes. After adding 3.5 ml of fresh media, determine the cell concentration using a hemocytometer.
  3. Add the cells to the six well plate at the following concentration:  
    • 500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips => ~35-40 cells/field-of-view at 63x, with ~20% transfected
    • 1 million cells/well for subsequent parachuting of cells onto coverslips
  4. Rock the plate sideways to evenly distribute the cells.
  5. Culture the cells overnight at 37oC.


Transfection
  1. Thaw the DNA to be used for transfection.
  2. Set up a reaction for each well in a microcentifuge tube:
    • Add 4.0 µg of DNA to 200 µl of serum-free media. Vortex the tube for 1-2 seconds to mix.
    • Add 10 µl of the Fugene-HD Transfection Reagent. Flick the tube 15-20 times to mix.                                                                                                                                                                    Note: The Tranfection Reagent is ethanol-based and evaporates easily. Keep the Transfection Reagent in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use.
  3. Let the reaction sit for 20 min at Rm temp in the tissue culture hood, for the DNA-containing micelles to form.
  4. Add the total reaction volume of each microcentrifuge tube to its respective cell well, drop-by-drop. Shake the plate sideways to mix.
  5. Incubate the transfecting cells overnight at 37oC.


Paraformaldehyde Cell Fixation
  1. Aspirate the media. Wash the cells twice with non-sterile PBS, at 2mL/well. Note: Be careful not to knock the cells off the coverslips during the wash steps.
  2. Set up a batch reaction. For six wells, combine:
    • 1.2 ml 10X PBS
    • 3 ml 18% PFA
    • 10.8 ml ddH2O
  3. Add 2mL of reaction/well. Let the plate sit at Rm temp for 15 min. Note: Keep in the dark if transfected with a fluorophore.
  4. Aspirate. Wash once-twice with PBS and cover with 2 ml PBS/well.


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