小中大准备做内皮细胞的血管新生实验，想知道具体步骤及注意事项，比如是24孔板的话该加多少的胶，怎么加，注意哪些事项，需要哪些准备工作。感谢~
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你好，做实验之前需要做充足的准备，包括实验计划，方法，以及试剂设备等，实验步骤简单介绍一下，1.铺胶，2.种细胞，3培养，4拍照，5统计分析结果。还需要你自己查阅相关的资料已经corning的的说明书，
2.0 Coating Procedure
NOTE: Once gelled, Corning ® Matrigel ® matrix should be used immediately. We
recommend using Corning Matrigel matrix with a protein concentration of at least
10 mg/mL. The concentration of Corning Matrigel matrix is lot-specific and can be
found on the Certificate of Analysis. You can pre-screen matrigel lots and order lot-
specific vials of Corning Matrigel matrix with your preferred protein concentration
by contacting Corning.
2.1 Thaw Corning Matrigel matrix as recommended. Using cooled pipets, mix it to
homogeneity.
2.2 Keeping 24-well culture plates on ice, add 0.289 mL of chilled Corning
Matrigel matrix (10 mg/mL) per well. This quantity should be sufficient to
cover the entire growth surface easily. If Corning Matrigel matrix needs to be
diluted to 10 mg/mL, dilute with serum free medium.
2.3 Avoid air bubbles in Corning Matrigel matrix while pipetting the liquid into
each well. If air bubbles get trapped in the wells, centrifuge the plate at 300xg
for 10 minutes in a centrifuge that has been pre-cooled to 4°C.
2.4 Incubate plates at 37°C for 30-60 minutes.
2.5 The plates are now ready to use.
3.0 Endothelial Cell Tube Formation Assay
3.1 Prepare the endothelial tube formation plate as directed above.
3.2 Culture endothelial cells with desired endothelial cell medium to desired
confluence. For Corning HUVEC-2, HMVEC, and HMEC-1, 70-80%
confluence is recommended.
NOTE: Primary cells should be low passage number (e.g., HUVECs should not be
passaged more than 5 times).
3.3 Prepare endothelial cell suspensions by trypsinizing the cell monolayers and
resuspending the cells in culture medium with 5-10% serum or with your
desired angiogenesis promoters at 4x10 5 cells/mL when using Corning HUVEC-
2, HMVEC or HMEC-1 cells. Additional testing agents such as inhibitory
agents can be included at this step as well.
NOTE: Most endothelial cell media does not contain a sufficient concentration
of serum to deactivate trypsin. The use of a trypsin neutralizing solution is
recommended.
3.4 Add 300 μl of the cell suspension (1.2x10 5 cells of Corning HUVEC-2,
HMVEC, or HMEC-1) to each well.
3.5 Incubate the angiogenesis assay plate for 16-18 hours at 37°C, 5% CO 2
atmosphere.
注意事项:1. 铺胶要尽量保持在低温条件下完成，比如在96孔板下放上预冷的冰盒，速度要尽可能快，以保证胶面平以利于拍照成像。2. 细胞计数时一定要尽量严格，以保证各孔细胞密度均匀。3. 种细胞时尽量从孔的中间位置加入，力度柔和，速度尽量慢，以免冲击力太大破坏胶面。
希望能对你有帮助
如实验中遇到其他问题随时讨论，祝实验顺利。
