HEK 293T Tissue Culture Protocols

Cell Splitting

Materials and Reagents:

• HEK293T Media
• Phosphate Buffered Saline (PBS)
• Trypsin
• Biosafety Cabinet
• 10 and 5 mL sterile pipets
• Vacuum apparatus w/ autoclaved Pasteur pipets.
• T-75 cell culture flask (or 60 mL petri dish for smaller culture).

Steps:

1. Prepare Biosafety Cabinet

   1. Turn off UV light.
   2. Turn on venting and lighting.
   3. Open cabinet door to working position (see marks on the cabinet door).
   4. Spray down inside surfaces of cabinet with 70% ethanol and wipe down.

2. Bring materials and reagents into Biosafety Cabinet.

   1. Spray with 70% ethanol and wipe down all surfaces before placing them in the cabinet.
   2. Be careful to use sterile reagents, or to filter non-sterile reagents.
3. Use vacuum apparatus and a Pasteur pipet to aspirate old media.

4. Rinse with 3-5 mL PBS (2 mL in 60 mL petri dish).

   1. Pipet onto the side of the dish/flask rather than onto the surface that the cells are laying on to avoid power-washing them away.
5. Aspirate PBS.

6. Add 3mL Trypsin (1 mL in 60 mL petri dish)

   1. This time you actually do want to apply the trypsin to the cell-containing surface.
7. Let sit for 3-5 minutes, until all the cells are loosely floating (can be seen in a microscope).

8. Add 7 mL media (2 mL media in 60 mL petri dish) and mix until cells are consistently spread through the solution.

   1. Repeatedly suck suspension into pipet then expel into the flask, thus breaking up cell chains and groups.
9. Pipet a calculated aliquot of cell suspension into the new, unused 75 cm^2 cell culture flask (or 60 mL petri dish).
10. Q.S. to 25 mL media + cells in new flask (or dish).
11. Aspirate unused cell solution and discard used cultureware.
12. Mark new flask with cell line, PD (population doubling; see Basic Cell Passage/Culture for how to calculate), split ratio (1:20-1:2), initials, and date.
13. Store flask in humidified incubator set to 37^oC and 5% CO₂,

14. Clean up Biosafety Cabinet and return reagents/media to the fridge.

Cell Freezing

Materials and Reagents:

• HEK293T Media
• Phosphate Buffered Saline (PBS)
• Trypsin
• Biosafety Cabinet
• 10 and 5 mL sterile pipets
• Vacuum apparatus w/ autoclaved Pasteur pipets.
• 75 cm^2 cell culture flask (or 60 mL petri dish for smaller culture).

• DMSO
• 2 mL freezing tubes
• Freezing Container

Steps:

1. Follow steps 1-7 in aforementioned procedure.

2. Prepare freezing media.

   1. 5% DMSO: mix 10 mL media with 0.5 mL DMSO.

3. Filter freezing media.

   1. Using a syringe to run your 5% DMSO solution through a filter.
   2. It is helpful to use a pipet to place the solution in the tail end of the syringe connected to the filter, then to insert the plunger and use it to propel the solution through.
   3. Do not retract plunger after filtering while the filter is still attached, as this will pull impurities back into the syringe. First disconnect the filter, then retract the plunger, then reconnect syringe and filter.
4. Chill freezing media for 3-5 minutes.

5. Add 7 mL media to trypsinized solution. Mix.

   1. Repeatedly suck solution into pipet, and then spray back into the flask, thus breaking up cell chains and groups.
6. Split into T-75 flask at a 1:20 ratio (i.e. take 0.5 mL of your 10 mL solution).

7. Q.S. to 25 mL (media + cell suspension).

   1. Mark new flask with cell line, PD, split ratio, initials, and date.

8. Centrifuge remaining cells out of trypsinized solution.

   1. Pipet trypsinized solution into 15 mL tube.
   2. Place in centrifuge with an appropriately sized counter-weight.
   3. Set centrifuge for 3 minutes (with the centrifuge we have in the tissue culture room right now, wind the timer past 3 minutes and then move it back, otherwise the centrifuge will never stop).

9. Mix cells with chilled freeze media.

   1. Repeatedly suck solution into pipet, and then spray back into the flask, thus breaking up cell chains and groups.

10. Split cell solution into six 2 mL freezing tubes, mark.

    1. Mark new flask with cell line, PD (population doubling; see Basic Cell Passage/Culture for how to calculate), split ratio (1:20, etc.), initials, and date.
11. Place freezing tubes in Freezing Container.
12. Place Freezing Container in -80°C for 4-24 hours.

13. Move freezing tubes to liquid nitrogen tank.

    1. Remember to work quickly while in the nitrogen tank to avoid warming other samples.