When fusing cells in suspension or monolayer cell systems, the treatment of cells with PEG should be short to minimize cell death. Usually, a 24 h recovery period is required for the cells before using selective media.
Operation method
Program 27.10 Cell Hybridization
Materials and Instruments
PEG Move PEG 1000 (Merck): For more product details, please visit Aladdin Scientific website.
Complete medium Serum-free medium NaOH
Petri dishes General purpose containers
(a) High-pressure treatment of PEG to liquefy and sterilize it.
(b) Cool the PEG to 37°C and mix with an equal volume of serum-free medium preheated to 37°C.
(c) Adjust the pH to about 7.6~7.9 with 1.0 mol/L NaOH.
(d) Store the solution at 4°C for up to 2 weeks.
A. Fusion of monolayer cultured cells:
1. Inoculate two equal amounts of cells to be fused into 50 mm cell culture dishes. Inoculate each parental cell line at a concentration of 2. 5X105~2.5X 106 /dish .
2. Incubate the mixed cells overnight.
3. Warm the PEG-serum-free medium to 37°C, at which time the pH needs to be readjusted with NaOH.
4. Discard the original culture medium and wash the cells once with serum-free medium.
5. Add 3.0 ml of PEG solution to cover the monolayer of cells.
6. Time accurately for 1 min, after which the PEG is discarded and the monolayer is washed with 10 ml of serum-free medium and replaced with complete medium.
7. Incubate the cells overnight.
8. Add selective medium again.
B. Fusion of suspended cultured cells:
9. Mix the two parental cells ( 4X106 cells each) and centrifuge at 150 g for 5 min at room temperature. subsequent centrifugation and fusion are performed in 30 ml plastic universal containers or centrifuge tubes.
10. Make a suspension of the precipitate in 15 ml serum-free medium and centrifuge again.
11. Aspirate off all the medium.
12. Carefully add 1 ml of PEG solution using a pipette and gently blow to suspend the cells.
13. After 1 min, dilute the cell suspension with 9 ml of clear medium and aliquot into 2 universal containers or 1 centrifuge tube pre-filled with 15 ml of clear medium.
14. Centrifuge at 150 g for 5 min, discard the supernatant and resuspend the cells in complete medium.
15. Incubate overnight at 37°C.
16. Clone the cells in selective medium.