IP3測定法(NEN kit)

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

・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。