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细胞划痕实验(转)

作者:伟佛修缘   (离线)   [细胞生物学 ]   [细胞划痕 ]   时间:2016-09-18 17:28:51  向他请教

材料:

6 孔板(其他的也可以,但感觉6孔比较好,大小适中)

marker

直尺

20微升枪头(灭菌)

无血清培养基

PBS

准备:

所有能灭菌的器械都要灭菌,直尺和marker笔在操作前紫外照射30min(超净台内)

流程:

1。先用marker笔在6孔板背后,用直尺比着,均匀得划横线,大约每隔0.5~1cm一道,横穿过孔。每孔至少穿过5条线。

2。在空中加入约5X105个细胞,具体数量因细胞不同而不同,掌握为过夜能铺满。

3。第二天用枪头比着直尺,尽量垂至于背后的横线划痕,枪头要垂直,不能倾斜。

4。用PBS洗细胞3次,去处划下的细胞,加入无血清培养基。

5。放入375%co2培养箱,培养。按061224小时取样,拍照。

 

PS: 如果你连续监测24小时,你需要考虑到划痕缩小是细胞迁移和细胞繁殖共同作用的结果,而不是单纯的细胞迁移。如果你要单纯的考虑细胞迁移,你可以先用丝裂霉素处理一小时,抑制细胞的分裂,这样你的结果就是细胞迁移的作用了。

照片拍完之后,可以用image J 来测量划痕区域的像素定量比较细胞迁移的速度。

 

 

Gut. 2007 Jan;56(1):95-106. (sci Impact Factor 9.002 )

Thompson CC, Ashcroft FJ, Patel S, Saraga G, Vimalachandran D, Prime W, Campbell F, Dodson A, Jenkins RE, Lemoine NR, Crnogorac-Jurcevic T, Yin HL, Costello E.Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility. Gut. 2007 Jan;56(1):95-106. (sci Impact Factor 9.002 )

方法:In vitro wound-healing assay

Panc-1 and Suit-2 cells were treated with siRNA as described above. After incubation for 72 (CapG) or 48 (Gelsolin) h, the cells were removed by trypsinisation, counted and plated at 400000 cells/ml in 12-well dishes. Cells were incubated overnight yielding confluent monolayers for wounding. Wounds were made using a pipette tip and photographs taken immediately (time zero) and 12 or 16 h after wounding for Suit-2 and Panc-1 cells, respectively. The distance migrated by the cell monolayer to close the wounded area during this time period was measured. Results were expressed as a migration

index—that is, the distance migrated by siRNA treated (control or targeted) relative to the distance migrated by RISC-free control RNA treated cells. Experiments were carried out in triplicate and repeated at least five times.98620152.jpg


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