小中大
献给大家一个很详细的HUVEC培养protocol
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
