Our Thoughts (桀靜精微雷風相薄): 2011-08

Aug 31, 2011

08/31 Lab meeing-小嬿

1. to observe whether CTCF interact with EBV Rta by Immunoprecipitation assay
a) in TW01 inducible cell lines, CTCF and Flag-Rta can not be immunoprecipitated by each other. I also tried Flag beads, which are provided by Sigma, both of Flag-Luc and Flag-ER caught CTCF. I can not prove the interaction between CTCF and EBV Rta in TW01 inducible cell lines.
b) in co-transfected HEK293/293FT cell lines(Flag-Luc/ER/KR), CTCF can immunoprecipitate Flag-ER. However, the ratio of each plasmid is unfair, i will adjust the amount of these plasmids (may ignore Luc) and perform this experiment again.

2. to screen 12 putative target genes in CTCF/Flag-ChIP samples
利用Q-PCR的方式, 這12個genes無法在CTCF-ChIP的samples中被偵測到; 但利用一般PCR的方式, 則可偵測到JUN及H19(雖然band很微弱)
a) 在ChIP之前的sonication片段可能太短, 以至於所設計的這些將近500 bp PCR product的primer不能bind上DNA
b) ChIP的效率不好, 在抗體/磁珠/sample量的比例上該做些調整

Aug 29, 2011

8/24 Lab Meeting-ingrid

(1) 用IP將KR抓下，用WB(RL2)來確認O-GlcNAc變化程度；經更改IP流程，可以有效率以KR(660) from ICON將KR抓下(ERKV_Dox48h)，但是之後應用於ERKV_shLuc_Dox48h、ERKV_shOGTA1_Dox48h及ERKV_shMGEA5B2_Dox48h，卻效率不佳，以致於後來的RL2及PARP1皆無法辨認。目前持續改進IP流程中。Note:如果是要看KR蛋白質，建議Lysis buffer要加phosphotase inhibitor以避免KR的碎裂。

(2) 關於進行shRNA的Q-PCR，已完成實驗數據。但是分析時，應注意星號的標誌，正確為*p<0.05, **p<0.01, ***p<0.001(星號應水平標誌)。

(3) 經由計算細胞生長速率及考慮表現Luc蛋白質的程度，混合細胞得到TW01TetLuc pool :#2+ #8及TW05TetLuc pool:#1+ #3+ #5。

(4) 三種Flag Ab測試(Sigma F1804舊、Cell Signaling及Sigma F1804新)：認識Luc蛋白質在TW01TetLuc pool及TW05TetLuc pool的titer皆差不多，除了Cell Signaling背景比較髒。

Aug 23, 2011

8/17 Lab meeting-蔡小丸

1. 在HEK293細胞中共同表達KR, S634A/S636A, Luc及不同量的Pin1, 結果發現大量的Pin1會造成KR, S634A/S636A, Luc蛋白量的降低, 由於Luc已知不會與Pin1交互作用, 因此大量Pin1可能部份造成細胞general的影響了。
2. IP結果初步看到, KR及S634A/S636A會被sumo-1所修飾, Luc則沒有, S634A/S636A被sumo-1修飾的能力較差, 但仍需進一步實驗確認sumo-1修飾KR及S634A/S636A所扮演的角色。

Aug 11, 2011

8/10 Lab meeting

1. The trial of new medium KGM-2 ── comparing with medium DKSFM/EpiLife for culturing NP460hTert:

(1) Cells culturing in KGM-2 showed more like triangle shape rather than elongated morphology as seeding in DKSFM/EpiLife.

(2) Cells culturing in KGM-2 had slower proliferation rate (Doubling time:55.4hrs) than culturing in DKSFM/EpiLife medium (Doubling time:36hrs).

2.Different [Ca2+] treatments for 6 days in KGM-2 medium for NP460hTert

(1) Along the treatment, cells in 2 calcium free treatments showed poor adhesion to the dish (細胞沒有延伸或突觸等的貼附形態出現). *Mortality rate:32% and 35%.

(2) Cells in 0.1mM [Ca2+] showed good adhesion (細胞亦無層疊的現象) and had the lowest mortality rate (11%) among all treaments.

(3) Cells in 0.5mM,1mM and 1.5 mM Ca2+ showed the tendency to cluster and pile up together under high Ca2+ culture.(第四天後細胞堆疊明顯)

(4) Cells in 1.5mM Ca2+ showed the highest mortality rate (48%), almost 50% of the cells were dead at the measuring time. The mortality of 1mM Ca2+ treatment weas 24%, 0.5mM Ca2+ was27%.

(5) After trypisinization, cells didn't look bigger when counting.(通常要準備走上senescence或分化的細胞會變較大)

討論結果:

(1) KGM-2和DKSFM/EpiLife下細胞生長形態的照片因為seeding不均勻的關係，要再重新試驗新種下比較均勻的10cm dish中再照一次。(8/10已重照完畢)

(2) 不同鈣離子濃度的試驗因為細胞種的不均勻可再做一次試驗(等下禮拜一母盤有夠多的細胞)，而其中一個無鈣離子的對照組可改為0.05mM來試驗(0.1mM still a little bit high or not?!)

(3) 下禮拜可以使用不同濃度的retinoic acid treatment!(in medium DKSFM/EpiLife or KGM-2)，可試試看曹老師用serum來促進differentiation的方法!

Aug 10, 2011

08/10 Lab Meeting- 小夏

1. 在TW01TetER及TW05TetER上Serum starvation的條件: 6-well種50000-100000 incubate 48h，serum free組與complete medium組相比，serum free組細胞數少，細胞長得細細長長，在細胞數與細胞型態上有差異性。
補充：根據之前flow data (call cycle analysis) 看48小時的實驗，細胞6-well種100000不至於過滿，200000細胞會過滿。所以我偏愛100000 incubate 48h的condition。接著模仿MB教授的實驗方法看看serum (glucose順道一起看)對Rta-induced cellular senescence.的重要性。(Ref）
2. Establishment of TW05-EREV：
結果：
(1) PEG濃縮病毒可以提高virus infection的效率（5%至20%, combine TGFbeta可達到30%），而TGFbeta則效果不彰。
(2) 細胞先放大至6-cm dish，再種1000顆細胞至10 cm dish進行G418 selection的過程中細胞便死光。
討論：
從12-well放大不要一下就放到6-cm dish中，建議先至6-well較適當。而G418 selection的步驟應該要在細胞放大時就做，防止沒有病毒的細胞勢力擴大。
若現在手邊的細胞已經沒有綠光的存在了，便重新開始以濃縮病毒的方式感染細胞。

NP460hTERT in KGM2

由 sufang 在三, 08/10/2011 - 14:54 發表 Older Posts EBV infection EBV Rta NP460hTERT transient lytic

2011/08/10 Lab Meeting