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.
