アラキドン酸遊離測定法(石井功)

第二生化マニュアル目次
1. Materials


2. Assay steps(12 well dishの場合)

Cells on 12 well dish

Wash the cells once with 1.25 ml of 37℃-prewarmed serum-free medium

Add 0.75 ml of serum-free medium containing 0.1 μCi of [3H]Arachidonate

Overnight labeling in CO2-incubator

Wash the cells twice for 3 min each with Wash buffer (Tyroad Solution with HEPES containg 0.1% f.a.-free BSA)

Add 500 μl of Wash buffer and incubate for 3 min

Add 5 μl of Wash buffer containing agonists

Incubate for appropriate periods under 37℃

Aspirate 250 μl of the sup with a pipetman → count the radioactivity (for released radioactivity)

Add 250 μl of 2% Triton X-100

Solubilize the cells with shaking

Take 5 μl of the lysate → count the radioactivity (for incorporated radioactivity)

3. Points

第二生化マニュアル目次