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Cell Culture Media – Supplementation of Media and Media Handling
Guide to Supplementation of Cell Culture Media and Media Handling
All 1L liquid media are formulated without antibiotics, serum and L-Glutamine unless stated otherwise. Glutamine can be the most unstable component in liquid cell culture media. The 10x concentrated media does not contain antibiotics, serum, L-Glutamine or sodium bicarbonate whereas powder media contains L-Glutamine but not sodium bicarbonate. It is recommended to aseptically add the required amount of these supplements prior to use. Sodium bicarbonate can be added as a solid before sterilisation or as a sterile solution of 7.5% after sterilisation.
Guide to the addition of L-Glutamine (200 mM solution)
The below table refers to the appropriate amounts of L-Glutamine and Sodium Bicarbonate that needs to be added.
|
Medium (1 L) |
L-Glutamine (200mM/l) ml required |
Final Concentration of L-Glutamine (mg/L) |
|
DMEM |
20 . 0 |
584 |
|
DMEM / Ham F12 Standard |
20 . 0 |
365 |
|
Ham's F-10 |
5.0 |
146 |
|
Ham's F-12 |
5.0 |
146 |
|
MEM Iscove's |
20 . 0 |
584 |
|
Leibovitz L-15 |
10.3 |
300 |
|
M199 Earle's |
3.4 |
100 |
|
M199 Hank's |
3.4 |
100 |
|
McCoy's 5A |
7.5 |
219 |
|
MEM Alpha |
10.0 |
292 |
|
MEM Earle's |
10.0 |
292 |
|
MEM Hank's |
10.0 |
292 |
|
RPMI 1640 |
10.3 |
300 |
|
Waymouth's |
12.0 |
350 |
|
William's E |
10.0 |
292 |
Guide to the addition of Sodium Bicarbonate (7.5% solution or powder)
The table below demonstrates the amount of sodium bicarbonate that needs to be added.
|
Medium (1L) |
NaHCO3 (7.5% solution) ml required |
NaHCO3 (solid) g required |
Final concentration og NaCHO3 (mg/L) |
|
DMEM |
49.3 |
3.70 |
3700 |
|
DMEM / Ham F12 Standard |
32.5 |
2.438 |
2438 |
|
Ham's F-10 |
16.0 |
1.20 |
1200 |
|
Ham's F-12 |
15.7 |
1.176 |
1176 |
|
MEM Iscove's |
40.4 |
3.024 |
3024 |
|
Leibovitz L-15 |
- |
- |
- |
|
M199 Earle's |
29.3 |
2.20 |
2200 |
|
M199 Hank's |
4.7 |
.35 |
350 |
|
McCoy's 5A |
29.3 |
2.20 |
2200 |
|
MEM Alpha |
29.3 |
2.20 |
2200 |
|
MEM Earle's |
29.3 |
2.20 |
2200 |
|
MEM Hank's |
4.7 |
0.35 |
350 |
|
RPMI 1640 |
26.7 |
2.00 |
2000 |
|
Waymouth's |
29.9 |
2.24 |
2240 |
|
William's E |
29.3 |
2.20 |
2200 |
Preparation of 1 L Single Strength Media from 10 × Concentrate
- Measure out approx. 850 ml aseptically of sterile tissue culture grade water in a container of accurate size
- Stir and add 10 x medium
- Add a required amount of Sodium Bicarbonate
- Sera, antibiotics, L-Glutamine and various other supplement can be added to the solution at this stage
- Sterile 1 N NaOH or 1 N HCI can be used to adjust the pH of the solution to the desired value
- Tissue grade water can be used to adjust the volume of the solution to 1 L
- Completed medium should be refrigerated due to the limited shelf life
Preparation of 1 L Liquid Media from Powder Media
- 900 ml of cell culture water needs to be placed in a 1 L Erlenmeyer flask. Gentle stir and add the powder slowly, do not heat.
- Add Sodium Bicarbonate in a solid form, stir and adjust the solution with the water to 1L
- Control the pH and adjust with 1 N HCI to approximately 0.2 pH units below the value required
- Use 0.2 um filter to sterilise the medium
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