10.FLEXSTATIONによるカルシウム上昇反応-031230Yokomizo
試薬：
10 x HBSS
Gibco 14065-056 (Ca(+), Mg(+))
Fluo-3
Dojindo F023, 1 mg/tube を443 μL DMSOに溶解(2 mM stock)
Probenecid
Sigma P-8761 (Multidrug transporter inhibitor、Fluo-3の細胞外への輸送阻害)
20 % Pluoronic acid
Molecular probe P-3000 (Fluo-3の溶解性を高める)
1 M HEPES, pH 7.4
Buffer at least 100 ml per plate
1x HBSS, 2.5 mM Probenecid, 20 mM HEPES, pH 7.4 (for 100 ml)
10x HBSS 10 ml
Probenecid 71.4 mg (最初に1 mlのH2O + 1N NaOH 1-2 dropで溶解しておく)
1M HEPES 2 ml
Loading buffer (prepare just before the loading)(for 12 ml)
buffer 12 ml
2 mM Fluo-3 24 ul
20 % Pluoronic acid 24 ul
Serum 120 ul (final 1%)
以下はCHO細胞での実験例です。細胞数の至適化が必要と思われます。
付着細胞でのアッセイ:(細胞数少なくてすむ、感度やや低い。図1参照)
(前日) CHO細胞を4 x 10e4/well=100 μLでガラスボトムの96穴plate(Costar 3603)に巻き込み(4 x 10e5 /ml)
(当日)
0) Prewarm the machine at 37 degree (it takes 60 min)
1) Discard the culture media, tap the plate gently on kim towel
2) Apply 100 μL of loading buffer to each well
3) Incubate at 37 degree for 60 min in culture room
4) Wash the cells with 200 μL of buffer twice as 1)
5) Apply 150 μL of buffer to each well (alternative:100 μL buffer + 50 μL 4 x antagonist)
6) Prepare 4 x ligand (50 μL per well + dead volume 20 μL) in 96 well v-bottom dish (Corning 3894)
7) Prewarm the cells at 37 degree in the FlexStation (ca 10 min)
8) Assay
浮遊細胞でのアッセイ：(細胞数を多くすることで、感度を上げることが可能と考えられる。図2,3参照)
0) Prewarm the machine at 37 degree (it takes 60 min).
1) Label the cells with loading buffer at 37 degree for 60 min (example:1 x 10e7 cells / 2 ml buffer).
2) Wash the cells with 10 ml of buffer twice.
3) Count the cells and adjust to 2 x 10e6 / ml.
4) Apply 100 μL of the cell suspension to each well of 96 well-plate(Costar 3603).
5) Prepare 4 x ligand (50 μL per well + dead volume 20 μL) in 96 well v-bottom dish (Corning 3894).
6) Prewarm the cells at 37 degree in the FlexStation (ca 10 min)
7) Assay
図1. 付着細胞(40000/well)での実験例(CHO-BLT1細胞、阻害剤処理後、100 nM LTB4刺激、n=3, Ave+/- SD)
図2.浮遊細胞で細胞数を振った実験例(CHO細胞、ATP刺激, n=3, Ave+/- SD)
図3.上記実験の生データ(n=3, Ave+/- SD)