Endothelial cell growth medium (EGM) for HUVECs and DMEM preparation
Materials:
1. M-199 powder (Gibco, 31100-035): without HEPES, without NaHCO3 or DMEM/F12 powder (Gibco, 12400-024): with HEPES (15 mM), without NaHCO3 or DMEM powder high glucose (Gibco, 12800-017): without NaHCO3 or DMEM powder low glucose (Gibco, 31600-034): without NaHCO3 (glucose concentration: HG DMEM 4.5 g/L, LG DMEM 1 g/L). If there is no DMEM powder (HG), you can use DMEM powder (LG) plus glucose (7 g for 2 L HG DMEM).
2. Endothelial cell growth supplements (EGS, Merck, 02-102, 150 mg)
3. HEPES sodium salt (Sigma, MW:260.29)
4. NaHCO3 (Sigma)
5. Heparin sodium salt (Santa Cruz Biotechnology, sc-203075B)
6. Antibiotic-antimycotic (100X, Gibco, 15240-062), add before use.
7. FBS (Gibco 10270-106), add before use.
8. Autoclaved filters and bottles
1X medium filters with
0.2_files/image001.png){kind=small}
m PVDF
membrane
75 cm2 cell culture flask with flat cap
Procedure
1. Prepare 3L EGM without EGS
|
|
M-199 powder |
DMEM/F12 powder |
DMEM powder |
|
Medium powder |
2 packages |
3 packages |
2 packages |
|
HEPES (final 25 mM) |
13.0 g |
7.1 g |
|
|
NaHCO3 |
4.4 g |
3.6 g |
7.4 g |
|
Heparin |
0.2 g |
0.3 g |
|
|
H2O |
1.8 L |
2.8 L |
1.8 L |
Adjust PH to 7.2-7.3
Add H2O to the final volume 2L or 3L (DMEM/F12 powder), please use the volumetric flask instead of a graduated cylinder.
2. Sterilize the medium:
Filter
the medium by with 0.2m PVDF
membrane.
3. Add EGS:
Dissolve the 1 bottle (150 mg) EGS with 9 ml filtered EGM. Add 3 ml EGS into 1L EGM. The left EGS can be stock at -20��C, please label it clearly.
4. Add FBS (add before use)
For 1L EGM, add 110ml FBS to 10% EGM. This is enough for general use.
Our suggestion: for primary MAEC or "old" HUVECs (passage>6), increase the FBS into 20%.
5. Label the bottle clearly (medium type, your name, prepare date), and stock the EGM or DMEM at 4 ��C.
Label sample: EGM AA+EGS+10%FBS+, HL, 31/Mar/2022.
|
Medium containing
|
Label
|
|
EGM without AA, EGS. FBS
|
EGM AA- EGS- FBS-. Name, Date
|
|
EGM with AA, without EGS, FBS
|
EGM AA+ EGS- FBS-. Name, Date
|
|
EGM with AA, EGS, without FBS
|
EGM AA+ EGS+ FBS-. Name, Date
|
|
EGM with AA, EGS, FBS (10%)
|
EGM AA+ EGS+ 10%FBS+, Name, Date
|
|
DMEM High glucose with AA, FBS (10%)
|
HG DMEM AA+ 10%FBS+, Name, Date |
Updated on 31 Mar 2022 by HL