(1) Test preparation: a box of 200ul/1ml pipette tip (all the above items need to be autoclaved), alcohol cotton ball, waste liquid tank, test tube rack, micropipette, marker, petri dish, centrifuge tube.
(2) Discard the medium in the petri dish and wash twice with 1ml of PBS solution.
(3) Use pipette Tip to add 1ml Trypsin solution and digest for 1 minute (37°C, 5% CO2). Tap the wall of the culture flask with your hand, and observe that the cells have completely fallen off the wall.
(4) Add 1ml of serum-containing medium to stop the reaction.
(5) Use pipette Tip to blow and suck several times to disperse the cells completely.
(6) Put the culture solution into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.
(7) Resuspend the cells in culture medium, select 0.8X106 cells to add to a 35mm culture dish after cell count.
(8) Add the appropriate volume of complete culture solution to the centrifuge tube, mix the cells and gently add them to the culture dish to make them evenly distributed.
(9) Transfer the petri dish to a CO2 incubator for culture, and transfection the next day.
1) Preparation of transfection reagent
A. Add 400ul of denuclease water to the tube and shake for 10 seconds to dissolve the fat.
B. Store the reagent at -20 degrees Celsius after shaking, and shake before use.
2) Choose an appropriate mixing ratio (1:1－1:2/Liposome volume: DNA quality) to transfect cells. Add an appropriate volume of serum-free medium to a transfection tube. Add appropriate quality of MyoD or EGFP DNA, after shaking, add an appropriate volume of transfection reagent, and shake again.
5) Put the mixed solution at room temperature for 10-15 minutes.
6) Aspirate the medium in the culture plate and wash it once with PBS or serum-free medium.
7) Add the mixed solution and put the cells back in the incubator for one hour.
8) When the time is up, decide whether to remove the mixed solution according to the cell type, and then add complete medium to continue culturing for 24-48 hours.
Second passage of cells
1) 24 hours after transfection, observe the experimental results and record the expression of green fluorescent protein.
2) Pass the cells again, and re-pow the cells into the culture dish according to the appropriate density of 0.8X105 cells/35mm culture dish for immunostaining.
3) After incubating for 24 hours under normal conditions, fix it according to the staining requirements.
Post time: Jan-17-2023