Feb 06, 2019
Mammalian Cell Culture: Refreshing Media
- 1University of Arizona
- 481b Laboratory
Protocol Citation: Kenneth Schackart, Kattika Kaarj 2019. Mammalian Cell Culture: Refreshing Media. protocols.io https://dx.doi.org/10.17504/protocols.io.xqifmue
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: February 03, 2019
Last Modified: February 06, 2019
Protocol Integer ID: 19946
Abstract
This protocol explains how to refresh the media of cultured mammalian cells grown in a tissue culture flask.
Guidelines
Labcoat and gloves must be worn at all times.
Materials
- Gloves
- Cultured T-75 flask
- Cultured T-25 flask
- Serological pipette with tips
- Dulbecco's Phosphate Buffered Saline (DPBS)
- Cell culture media (e.g. DMEM:F12, EMEM, etc.)
Assessing Culture Health
Assessing Culture Health
Look at cells cultured in T-75 flask using the light microscope.
Note
Separate protocol for microscope use provided at microscope station and online.
Take image of the cells.
Iclude this image in the Results section of the lab report.
Include an assessment of culture health based on cell morphology in Discussion section.
Note
If you have been feeding too infrequently, you may see signs of poor health, such as cells rounding or looking less attached to the surface. You may also see the media turn yellowish orange, which is evidence of a pH change (most cell media have phenol red, a pH indicator)
This is a good time to check for contamination. Sometimes, contamination is immediately evident if culture media has become turbid. Or you may see bacteria moving when viewing under the microscope.
Contamination evidenced by media turbidity and color.
Refreshing media
Refreshing media
Using a serological pipette, remove old media from flask. Dispose of media in waste beaker. Dispose of pipette tip by sliding it back into its wrapper, and disposing in sharps box.
Note
To avoid contamination, avoid touching the pipette tip to anything. Holding the flask such that the lid is pointed up will make it easier to remove all the liquid.
Wash cells by pipetting 1 mL warmed DPBS into the flask.
Remove DPBS and dispose into waste beaker.
Repeat the above two steps for a total of 2 washes.
Pipette 4 mL warmed cell culture media into flask.
Incubate
Incubate
Spray flask generously with 70% Ethanol solution before placing in CO2 incubator.
Note
This will be performed by T.A.