蛋白活性實(shí)驗(yàn)-TNF-a活性蛋白
蛋白活性實(shí)驗(yàn)-TNF-a活性蛋白
TNF-a是內(nèi)源性的熱原，能夠引起發(fā)熱、凋亡細(xì)胞死亡、炎癥和抑制腫瘤發(fā)生。據(jù)報(bào)道，TNF-a可以抑制A549細(xì)胞的增殖和誘導(dǎo)凋亡，在刺激后細(xì)胞上清液中IL-1的濃度會(huì)增加。
實(shí)驗(yàn)方法：因此,A549細(xì)胞在DMEM孵化TNFa(1 ng / mL,10 ng / mL)2 h,4 h,8 h,24小時(shí)、48小時(shí),然后細(xì)胞被倒置顯微鏡觀察,IL-1β被ELISA檢測(cè)細(xì)胞上清液中。
實(shí)驗(yàn)結(jié)果：用TNF-a (10ng/mL)孵育72h后觀察細(xì)胞凋亡(見圖一）。
A B
Figure 1. Effect of TNF-a on A549 cells.
(A) A549 cells cultured in DMEM, stimulated with 10ng/mL TNF-a for 72h;
(B) (B)Unstimulated A549 cells cultured in DMEM for 72h.
與TNF-a孵化后(10 ng / mL)8 h,IL-1β顯著增加的細(xì)胞上清液（見表一）。
Sample (cell supernatant of A549 cells) | O.D. value | Corrected | Concentration of IL-1β (ng/mL) |
stimulated with TNF-β (10ng/mL) | 1.210 | 1.046 | 52.0 |
unstimulated | 0.187 | 0.023 | 4.9 |
Table1.Effect of TNF-β on A549 cells by ELISA.
STD (ng/mL) | O.D. value | Corrected |
150 | 3.363 | 3.199 |
75 | 1.630 | 1.466 |
37.5 | 0.974 | 0.810 |
18.75 | 0.442 | 0.278 |
9.375 | 0.271 | 0.107 |
4.6875 | 0.180 | 0.016 |
2.34375 | 0.168 | 0.004 |
The standard curve was demonstrated as follows：