Download Cell Culture and Transfection of Expression

Cell Culture and Transfection of Expression Vectors
Coat Coverslip

Gelatin can also be used for the culture of some cell types including glial cells
1. Dissolve 100 mg gelatin in 100 ml water (triple glass distilled or RO).
2. Autoclave to sterilize.
3. While hot, thoroughly mix gelatin solution.
4. Add enough solution to pool over surface of sterile glass coverslip.
5. Chill for 2-24 hours at 4oC.
6. Remove gelatin by aspiration and add sterile water.
7. Dishes can be stored for up to one week at 4oC.

Remove water immediately before use for cell cultureglass coverslips that had been
coated with fibronectin (1 h, 37°C) and blocked with 2% bovine serum albumin
(BSA) (30 min, 37°C).
Seed Cells
Cells were seeded onto 35-mm plates at 1.5 × 105/plate for time-lapse videomicroscopy
experiments.
Cells were seeded onto 12-mm glass coverslips at 2 × 104 for immunofluorescence cell
staining experiments.
Cells were seeded onto 10-cm plates at 2 × 106 for coimmunoprecipitations and kinase
assays.
Culture Cells
The cells were grown at 37°C and 10% CO2 in complete growth media: sodium
bicarbonate-buffered Dulbecco's Modified Eagle's Medium (DME) with 10% fetal bovine
serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids,
100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma Chemical).
Transfection(Lipofectamine-Invitrogen)
1.Incubate the cells at 37oC in a CO2 incubator until the cells are 70-80% confluent. This
will usually take 18-24 h.
2.Prepare the following solutions in 12 x 75 mm sterile tubes:
Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free
IMDM (containing nonessential amino acids).
Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl
serum-free IMDM.
3.Combine the two solutions, mix gently, and incubate at room temperature for 15-45
min. The solution may appear cloudy, however this will not impede the transfection.
4.Wash the cells once with 2 ml serum-free IMDM.
5.For each transfection, add 750 μl serum-free IMDM to each tube containing the lipidDNA complexes. Do not add antibacterial agents to media during transfection. Mix
gently and overlay the diluted complex solution onto the washed cells.
6.Incubate the cells for 5 h at 37E C in a CO2 incubator.
7.Add 1.5 ml IMDM with 20% FBS without removing the transfection mixture. If
toxicity is a problem, remove the transfection mixture and replace with normal growth
medium.
8.Replace medium at 18-24 h following start of transfection.
9.Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on
cell type and promoter activity.
Fix Cells
The cells were fixed with 4% formaldehyde in 0.1 M sodium phosphate (pH 7.0) 36 hr
after the transfection.
The nuclei of some fixed cells were stained with 1 µg/ml of 4',6-diamidino-2phenylindole-2HCl (DAPI) in 5 mM phosphate-buffered 0.9% (w/v) saline, pH 7.4
(PBS).
Micoroscope
GFP (excitation 450–490 nm, emission 515–565 nm)
DsRed (excitation 540–552, emission 575 nm)
DAPI (excitation 359–371, emission 397–490 nm)