6.IP3 測定法（NEN kit）（石井功）
1. Cell Preparation
Confluent cells on 75 cm2 flask
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Wash the cells once with 30 ml of PBS(-) containing 1 mM EDTA
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Add 20 ml of PBS(-) containing 1 mM EDTA and incubate in CO2-incubator (cell remove)
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Collect the cells by pipeting to 50 ml Falcon tube
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cfg 1200rpm for 10 min at 4℃
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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)
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Cell Counting
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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
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Incubate at 37℃ for aporopriate periods
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Add 200 μl of ice-cold 100 (w/v)% TCA (reaction stop)
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Vortex throughly and stand on ice for 15 min
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cfg 15,000rpm for 1 min at 4℃
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Take all the sup carefully to orange-capped Corning tube
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Stand at room temp. for 15 min
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Add 2 ml of freshly-prepared TCTFE-trioctylamine (3:1) and vortex vigorously for 15 seconds
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Stand at room temp. for 3 min
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Take all the upper phase to Eppendorf Tube
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Store on ice or 4℃
3. Assay steps
Prepare the mixture (total, blank, standards, samples)
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1 hour binding at 4℃ in foam racks
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cfg 3,000rpm for 10 min at 4℃ in foam racks
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Discard the sup completely by shaking sharply downward
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Add 50 μl of 0.15 M NaOH
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Vortex for 3 seconds
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Stand at room temp. for 10 min
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Vortex again for 5 seconds
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Add 5 ml of Atomlight to each tubes
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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。