Calcium Phosphate transfection of 293T cells

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.