TITLE: Protocol for the growth and maintenance of mammalian cell lines
AUTHORS: Agnes Roczniak-Ferguson, Shawn M. Ferguson

[DESCRIPTION]
This protocol details about the growth and maintenance of mammalian cell lines.

[STEPS]
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
1. Remove 1x PBS, trypsin, and media (DMEM+10%FBS+1%pen/strep supplement (Gibco) from the fridge and warm it up in a 37 °C water bath for 10 min.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
2. Remove containers from water bath and disinfect with 70% EtOH before placing in cell culture hood.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
3. Take the existing cells in the 75 17152 cell culture flask in the incubator and place under hood after observing under microscope to ensure that cells are near confluent.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
4. Attach a sterile Pasteur pipette to the vacuum tube so that the media can be aspirated from the flask by tilting the container so that the media is at front facing left corner where the pipette tip is. 
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
5. Wash cells with 10 mL of PBS added to the bottom most surface away from the cells then place flask down horizontally and gently move side to side to rinse the cells.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
6. Aspirate again.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
7. Add 3 mL of pre-warmed trypsin to cover the cells.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
8. Incubate for 5 min or until the cells have released from the flask.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
9. Add 5 mL of fresh media.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
10. Collect cell suspension from the flask and wash the side possessing the cells several times before transferring solution to 15 ml conical tube.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
11. Centrifuge the conical tube for 2.5 minutes at 100 x g.  
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
12. Aspirate to leave behind just the cell pellet and a little bit of media to cover the cell pellet.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
13. Resuspend in 10 mL of media and pipette up and down to disperse cell clumps. Avoid the formation of bubbles.
SECTION: The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
14. Obtain a new flask and place 12 mL of fresh media into it and before transferring 1 mL (1:10) of the cell suspension into it. Or alternatively distribute the desired number of cells (counted with hemocytometer or other cell counting device) to the appropriate cell culture dish/flask for downstream experiments.
SECTION: Phosphate Buffered Saline (PBS) buffer
15. 
 

SECTION: Phosphate Buffered Saline (PBS) buffer
16. Place 3.5 L Milli-Q water in a 4L beaker and add chemicals.  Stir on a stir plate until dissolved.
SECTION: Phosphate Buffered Saline (PBS) buffer
17. Once dissolved, check pH and adjust to pH 7.4.
SECTION: Phosphate Buffered Saline (PBS) buffer
18. Pour the solution into a 4L graduated cylinder and adjust volume to 4 L with water.
SECTION: PBS for Cell Culture
19. Make 2 L of 1x PBS pH 7.4:  200 mL of 10x PBS pH 7.4 + 1800 mL of H2O
SECTION: PBS for Cell Culture
20. Decant into 250 ml bottles and autoclave 40 min on liquid cycle.