in vitroでの血管新生アッセイにおける3次元(3D)は2Dの文化を使って達成できるよりも生体内で実際の環境にかなり近いですモデルを提供する成長コンセンサスがある。それは、優れた3Dシステムは再現性、及び血管新生の主要なステップのいくつかを模倣することができるはず明らかである。いくつかの以前の3Dアッセイが開発されているが、これらの多くは、いずれかを使用して、ハードを取得するため?…
Materials
Material Name
Typ
Company
Catalogue Number
Comment
Cytodex-3 Beads
Reagent
Amersham Pharmacia
17-0485-01
10 g/bottle.
1. 0.5 g of dry beads are hydrated and swollen in 50 mL PBS (pH=7.4) for at least 3 hours at RT.
Use a 50 mL tube and place it on the rocker.
2. Let the beads settle down (~ 15 min). Discard the supernatant and wash the beads for a few minutes in fresh PBS (50 mL).
3. Discard the PBS and replace with fresh PBS:
25 mL -> 20 mg/mL => 60000 beads/mL or
50 mL -> 10 mg/mL => 30000 beads/ mL
4. Place the bead suspension in a siliconized glass bottle (Windshield Wiper or Sigmacote).
5. Sterilize the beads by autoclaving for 15 min at 115C.
6. Store it at 4C.
Aprotinin
Reagent
Sigma
A-1153
10mg/bottle. Reconstitute lyophilized aprotinin at 4 U/mL in DI water. Sterile filter. Make aliquots of 1 mL each. Store at -20C.
Fibrinogen Type I
Reagent
Sigma
F-8630
1g. Dissolve 2 mg/mL fibrinogen in DPBS
Note clottable protein % and adjust accordingly
Heat in a 37C-water bath to dissolve the fibrinogen. Mix by inverting the tube. Do not vortex.
Sterile filter through 0.22 um
Thrombin
Reagent
Sigma
T-3399
22 mg=1000 units. Reconstitute in sterile water at 50 U/mL. Make aliquots of 0.5 mL each. Store at -20C.
Nakatsu, M. N., Davis, J., Hughes, C. C. Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis. J. Vis. Exp. (3), e186, doi:10.3791/186 (2007).