「DAG study I am doing recently」の編集履歴（バックアップ）一覧に戻る
19/Aug/2010
MDCK cells were incubated with 200 microM D609 for 12hour.
But cells died perfectly.
So I can not use long incubation with 200 microM D609.
I am gonna try 6hour.
18/Aug/2010
The results of yesterday experiment.
In fact, ISP-1 treated cells give annexin V fluorescence a little bit without Bc-PLC.
After incubation with 1.25units/ml Bc-PLC, in the case of 200 nM ISP-1, fluoresecence of annexin V increases little by little.
Same situation gonna happen in the case of 50 nM ISP-1.
But labelling with annexin V is very weaker than 200 nM.
This is the level I can ignore.
So I put 50nM IPS-1 treated cells in my paper.
17/Aug/2010
Today I gonna do the following experiments.
13/Aut/2010, I did 12.5 unit/ml BcPLC application experiment.
I want to confirm that ISP-1 treated cells do not have cell damages during incubation with 12.5 unit/ml Bc-PLC using anexin V.
And I want to confirm whethere DAG produced by Bc-PLC is same between tISP-1 treated cells and normal cells.
13/Aug/2010
I did several important expriments.
1. whether ZS2, ZS2/SMS1 and ZS2/SMS2 have DAG on the outer leaflet of PM.
Answer: Every cells was stained with DAG probe at the same level.
This shows that PC-PLC activity is different from SMS1 and SMS2.
2. whether D609 diminishes the outer DAG amount in MDCK cells.
Answer: at 6hour DAG diminished however 24 hour incubation recovered the DAG? What happen??
Anyhow I am gonna take reproducibility.
3. Glutamte applies MDCK cells expressing mGluR5.
20 microM glutamate was added to this MDCK cells.
However there is no difference between not expressed cells and expressed cells.
This data shows that DAG does not flop to the outer leaflet of the PM.
4. 1.25 unit/ml Bc-PLC was added to the MDCK cells treated with 50 and 200 nM ISP-1.
Flip occured!
10/Aug/2010
Here is most important paper now for me.
[[D609>http://breast-cancer-research.com/content/12/3/R27]]
[[D609-1>http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2M-40963W3-3&_user=107951&_coverDate=05%2F31%2F2000&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000008318&_version=1&_urlVersion=0&_userid=107951&md5=a6073cf38d66b16f7febabff9d2bb193]]
In the experiment of 10/Aug/2010, we could not see difference in fluorescence between D609 24hour incubated MDCK cells and intact MDCK cell.
So I am going to do this reproducibility again.
And then I am gonna start ISP1 incubation again.
06/Aug/2010
I am gonna do ISP-1 treatment expeiment.
I am gonna divide MDCK cells, which exposed 200nM and 50nM ISP-1 for observation and biochemistry expeiments.
So I gonna prepare six 3.5mm well dishs for each.
And I gonna do the experiment in which I want to see the effect of D609 on the receptor signalling.
Because it is known that D609 causes the internalization of receptors from plasma membrane.
So I prepare MDCK cells expressing inner DAG probe in three dishes.
03/Aug/2010
I got mGluR-RFP from Matsuura-kun.
I can express this DNA whatever cells I want.
I am gonna express this DNA CHO cells and MDCK cells.
Today's result.
I looked through the samples I prepared yesterday.
Now I found out that DAG exists in static state.
Based on this result, I need to plan the expriments.
Basically, this expreiment follows the experiment of CHO cells.
02/Aug/2010
I am gonna do the experiment as follows.
1. no probe, probe only, ATP, ATP+R59949, ATP+U73122, PC-PLC.
2. MDCK cell growth with or without DAG probe.
01/Aug/2010
I am doing the experiments in which 10 microM R59949 incubation is done upon 100 microM ATP addition.
I expect that more DAG comes to outer leaflet of PM than without R59949.
10 min R59949 incubation and then ATP stimulation containing R59949.
2 min incubation with DAG probe.
