08452 30 40 30
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
Want to Know More About Media and Media Handling?
Contact us today, our team will be happy to help with any questions you may have.
|
To Find Out More |