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Transfection methods that work for WIF-B cells

  1. Recombinant adenovirus:

The following protocol is used for WIF-B cells grown on 1x1 cm coverslips and housed in 6 cm Falcon dishes.

  1. Remove medium. Rinse with 2-5 ml of sterile Hanks balanced salt solution or PBS++.
  2. Virus sup application:
    1. for 200 ml of viral sup (try this vol first, a 1:10 dilution may also be appropirate to test) suction dish and around coverslip edges with a sterile pasteur pipet to reduce surface tension. Process a max of 3 coverslips at a time, the cells will dry out if left too long before addition of medium. Apply 200ml of virus sup to coverslip.
    2. for 1-2 ml of virus sup ( use this vol if 200 ml does not give expression). Apply 1-2 ml of virus sup to dish.
    3. for control uninfected, apply appropriate vol of 10% FCS DMEM.
  3. Alternatively, purified virus may be used for infecting WIF-B cells on coverslips (estimate is ~2 x 106 cells/ coverslip)
    1. Dilute virus to 2.5-0.5 x 1010 virus particles/ml (determined by OD260) in Optimem (Gibco BRL cat no. 51985-034).
    2. Apply 200ml of diluted virus (=1-5 x 109 virus particles).
    3. These values are intended as a guide and not absolute values since multiple factors may impact the amount of virus required for expression of the exogenous protein including the method for generating the recombinant adenovirus, the purity of the virus, and method of purification. The range indicated in "b" above reflects the range in virus purity that we have observed.
  4. Incubate at 37 C for
    1. 30 min for 200 ml
    2. 60 min okay for larger volumes. Sups have been left overnight, but viral toxicity was observed.
  5. Remove virus, wash once as in step 2, then replenish cells with medium and incubate at 37 C for 24 to 48 hrs. Test for expression by IMF or western blotting.
  1. Stable Transformation:

For a complete protocol describing selection of clones stably expressing your gene of interest , please contact Gudrin Ihkre