第二生化マニュアル目次
1. Cell Preparation

Confluent cells on 75 cm2 flask
↓
Wash the cells once with 30 ml of PBS(-) containing 1 mM EDTA
↓
Add 20 ml of PBS(-) containing 1 mM EDTA and incubate in CO2-incubator (cell remove)
↓
Collect the cells by pipeting to 50 ml Falcon tube
↓
cfg 1200rpm for 10 min at 4℃
↓
Resuspend the cells in 20 ml of ice-cold Suspension Buffer (Tyroad Solution with 10 mM HEPES(pH7.4) containing 0.1% fatty acid-free BSA)
↓
Cell Counting
↓
Resuspend the cells in Suspension Buffer to get a cell density of 2.5x105〜1x106/ml　


2. Cell Stimulation

Add 200μl of Suspension Buffer containing agonists to 800μl of Cell Suspension
↓
Incubate at 37℃ for aporopriate periods
↓
Add 200 μl of ice-cold 100 (w/v)% TCA (reaction stop)
↓
Vortex throughly and stand on ice for 15 min
↓
cfg 15,000rpm for 1 min at 4℃
↓
Take all the sup carefully to orange-capped Corning tube
↓
Stand at room temp. for 15 min
↓
Add 2 ml of freshly-prepared TCTFE-trioctylamine (3:1) and vortex vigorously for 15 seconds
↓
Stand at room temp. for 3 min
↓
Take all the upper phase to Eppendorf Tube
↓
Store on ice or 4℃
3. Assay steps

Prepare the mixture (total, blank, standards, samples)
↓
1 hour binding at 4℃ in foam racks
↓
cfg 3,000rpm for 10 min at 4℃ in foam racks
↓
Discard the sup completely by shaking sharply downward
↓
Add 50 μl of 0.15 M NaOH
↓
Vortex for 3 seconds
↓
Stand at room temp. for 10 min
↓
Vortex again for 5 seconds
↓
Add 5 ml of Atomlight to each tubes
↓
Count the radioactivity

4. Points

・各操作を添付のマニュアル通りにきちんと行えば、かなり簡単に測定できる。
・検量域にサンプルがうまくのるように、細胞数を調整する。
・マニュアルにはのっていないが、細胞を加えないSuspension BufferのみによるBack Groundの測定による補正が必要。
第二生化マニュアル目次