Protocol for primary cultures of HUVECs
I- Buffers and reagents
• Fetal calf serum (FCS) (Biosepra)
- Unfreeze the FCS
- Heat for 30 min. at + 56°C (decomplementation; bottle closed)
- Fractionate (25 ml) in sterile Falcon of 50 ml
- Freeze the fractions at – 20°C
• Phosphate Buffer Saline (PBS)
- 100 ml of PBS 10X without Ca++ and Mg++ (Life Technologies)
- 900 ml of distilled water
- 2 g of glucose
- Filtrate (0.22 μ), fractionate (50 ml) in sterile Falcon and freeze at –20 °C
• Collagenase (0.2 % in PBS)
- Dissolve 0.2 g of collagenase H from Clostridium Histolyticum (Roche) in 100 ml of PBS
- Mix during 10 min. in the dark (aluminium foil)
- Adjust the pH at 7.40 with NaOH 1N
- Filtrate (0.22 μ)
- Fractionate (10 ml) and freeze at –20°C
• Buffer for conservation and transport of umbilical cords
- PBS 50 ml
- Colimycine 1M 1 ml
- Penicilline G 1M 1 ml作者: newway 时间: 2012-5-29 11:56
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• Culture medium
M199 “stock”:
- M199 Earle 10X 100 ml
- Distilled water 900 ml
M199 “full”:
- Glutamine 0.2 M 1 ml
- HEPES 1 M 1.5 ml
- NaHCO3 7.5 % (w/v) 1.8 ml
- Penicilline/streptomycine (10000/10000) 1 ml
- FCS (heated at 56°C and centrifuged) 20 ml
- M199 “stock” up to 100 ml
Conservation at +4°C up to 1 week.
II- Cell culture
• The day before the manipulation:
- Coat the Petri dishes (35 mm; 7 dishes/cord) with 1 drop of fibronectin
- Bring at the maternity 3 recipients containing 50 ml of conservation buffer for cords
• The day of the manipulation:
- Fill up a recipient with ~ 500 ml of physiologic serum and maintain at 37 °C (“bain-marie”)
- Under the hood, prepare (for 1 cord): 作者: chuntian1983 时间: 2012-5-29 11:56
