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Cell Culture Media – Supplementation of Media and Media Handling

Guide to Supplementation of Cell Culture Media and Media Handling


Cell Cultures

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

Cell Cultures
  1. Measure out approx. 850 ml aseptically of sterile tissue culture grade water in a container of accurate size
  2. Stir and add 10 x medium
  3. Add a required amount of Sodium Bicarbonate
  4. Sera, antibiotics, L-Glutamine and various other supplement can be added to the solution at this stage
  5. Sterile 1 N NaOH or 1 N HCI can be used to adjust the pH of the solution to the desired value
  6. Tissue grade water can be used to adjust the volume of the solution to 1 L
  7. Completed medium should be refrigerated due to the limited shelf life

Preparation of 1 L Liquid Media from Powder Media

  1. 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.
  2. Add Sodium Bicarbonate in a solid form, stir and adjust the solution with the water to 1L
  3. Control the pH and adjust with 1 N HCI to approximately 0.2 pH units below the value required
  4. Use 0.2 um filter to sterilise the medium


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