Jun 05, 2024
Protocol for the growth and maintenance of mammalian cell lines
- Agnes Roczniak-Ferguson1,2,
- Shawn M. Ferguson1,2
- 1Departments of Neuroscience and of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Agnes Roczniak-Ferguson, Shawn M. Ferguson 2024. Protocol for the growth and maintenance of mammalian cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rrq4g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2024
Last Modified: June 05, 2024
Protocol Integer ID: 101277
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol details about the growth and maintenance of mammalian cell lines.
Materials
Phosphate Buffered Saline (PBS) buffer
| A | B | C | D | |
| Reagents | 1 liter of 1X | 4 liter of 10X | Final concentration (mM) | |
| Potassium Phosphate monobasic (KH2PO4) | 0.144g | 5.76g | 1.1 mM | |
| Sodium chloride (NaCl) | 9g | 360g | 155.2 | |
| Sodium Phosphate dibasic (Na2HPO4) | 0.421g | 16.842g | 3 |
The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
The following protocol was written for HeLa cells but can be adapted to many other standard mammalian cell lines.
15m
15m
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 00:10:00 .
10m
Remove containers from water bath and disinfect with 70% EtOH before placing in cell culture hood.
Take the existing cells in the 75 µL 2 cell culture flask in the incubator and place under hood after observing under microscope to ensure that cells are near confluent.
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.
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.
Aspirate again.
Add 3 mL of pre-warmed trypsin to cover the cells.
Incubate for 00:05:00 or until the cells have released from the flask.
5m
Add 5 mL of fresh media.
Collect cell suspension from the flask and wash the side possessing the cells several times before transferring solution to 15 ml conical tube.
Centrifuge the conical tube for 2.5 minutes at 100 x g .
Aspirate to leave behind just the cell pellet and a little bit of media to cover the cell pellet.
Resuspend in 10 mL of media and pipette up and down to disperse cell clumps. Avoid the formation of bubbles.
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.
Phosphate Buffered Saline (PBS) buffer
Phosphate Buffered Saline (PBS) buffer
| A | B | C | D | |
| Reagents | 1 liter of 1X | 4 liter of 10X | Final concentration (mM) | |
| Potassium Phosphate monobasic (KH2PO4) | 0.144g | 5.76g | 1.1 mM | |
| Sodium chloride (NaCl) | 9g | 360g | 155.2 | |
| Sodium Phosphate dibasic (Na2HPO4) | 0.421g | 16.842g | 3 |
Place 3.5 L Milli-Q water in a 4L beaker and add chemicals. Stir on a stir plate until dissolved.
Once dissolved, check pH and adjust to 7.4 .
Pour the solution into a 4L graduated cylinder and adjust volume to 4 L with water.
PBS for Cell Culture
PBS for Cell Culture
40m
40m
Make 2 L of 1x PBS 7.4 : 200 mL of 10x PBS 7.4 + 1800 mL of H2O
Decant into 250 ml bottles and autoclave 00:40:00 on liquid cycle.
40m