Materials
• Complete medium: DMEM(high glucose) supplemented w/ 10% BCS and Gln (Pen/Streptmycin: optional)
• Transfection reagents:
i) 2M CaCl2: 2M in H2O, filter
sterile, store at 4°C
ii) 2xHBS: 8.0g NaCl, 0.37g KCl, 201mg Na2HPO4•7H2O,
(1.0g glucose), 5.0g HEPES/500ml (adjust pH to 7.05 with NaOH and filter
sterile, store at 4°C)
• DNA
• Chloroquine stock, 25 mM in PBS (freeze
in vials)
• (Optimem supplemented w/ ITS: optional)
Procedure
The following protocol is for 10 cm dish
(medium: 10ml). If you use 6-well or 12-well plates, total volume of the medium
should be 3ml and 1.5ml, respectively, and decrease the amount of each reagent
accordingly.
1. Plate cells the night before to give
60-80% confluence at the day of transfection.
2. One hour prior to the transfection,
change to medium containing 25µM chloroquine (from x1000 stock in PBS, stored
at -20°C). Volume should be 10 ml per dish. (Chloroquine can be omitted, but
increases efficiency about x2)
3. Add 10µg DNA and ddH2O to
438 µl in 15-ml sterile tube, then add 62 µl 2M CaCl2 to a final
volume of 500 µl. When you are ready, add 500 µl of 2xHBS dropwise while gently
mixing. Add this mixture directly to the cells dropwise through the medium. Do
this within 1-2 min after adding 2xHBS. You will notice that the medium turns
to orange. Make sure you evenly sprinkle the droplet over the entire area.
4. Incubate for 7-11 h. Very fine,
dust-like precipitate visible. After incubation, rinse once and change to
medium without chloroquine (or to Optimem with ITS).
5. Culture supernatant, 2-6 days after
transfection. For secreted protein, you can change media once (at day 3 or 4,
save the collected sup) and give it another 3-4 days of culture. As long as the
cells are alive, they produce proteins. However, the time when the secretion
level reaches maximum varies among proteins.