胰岛素刺激3T3-L1细胞分化及样品制备
对于您实验的细胞,需要根据细胞本身的情况优化培养基和各项条件,如实际浓度、培养时间及其他实验相关参数。
以下是标准12孔培养板的3T3-L1细胞操作步骤。
试剂和培养基
・Serum-free culture medium
・Krebs Ringer Phosphate HEPES (KRPH) buffer at 37°C
(1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, 118 mM NaCl, 5 mM KCl, 30 mM Hepes, pH7.5)
・BSA (essentially fatty acid free and globulin free grade, use Sigma Ca. No. A0281 or equivalent)
・2-Deoxy-D-glucose (2DG) solution
・ Insulin solution
・ PBS(-)
・Phloretin or Cytochalasin B (or other glucose uptake inhibitor)
细胞样品制备
1) Differentiate 3T3-L1 cells to adipocytes in a 12-well culture plate.
2) Replace culture media with serum-free medium. Return to incubator for 6 hours.
3) Gently wash cells 3 times with 1.5 mL of warm KRPH buffer without BSA?.
4) Gently add 3 mL of warm KRPH buffer containing 2% BSA.
加入胰岛素
5) For this example, insulin is added to a final concentration of 1 μm .
6) Incubate cells for 18 min at 37°C.
加入2DG
7) For this example, 2DG is added to a final concentration of 1 mM.
8) Incubate cells at 37°C for 20 minutes.
9) Wash cells gently 3× with cooled PBS containing 200 μM Phloretin to inhibit further 2DG uptake.
细胞裂解及样品制备
10) Add 1.5 mL of 1× Sample Diluent Buffer.and lyse cells by sonication with a microtip sonicator.
11) Collect cell lysate to tube, and apply heat treatment at 80°C for 15 minutes.
Note: Do not add protease inhibitors to cell lysates.
Note: If an alternate method for cell lysis will be used, do not use NaOH as NaOH degrades 2DG6P.
12) Centrifuge lysates at 15000 x g for 20minutes at 4°C
13) Transfer cell lysate supernatants to a fresh tube. These cell lysate supernatants are your experimental samples.
Note: Supernatants can be stored at -20°C for later analysis.
检测细胞裂解液中的2 DG
14) Follow the procedure of [Section III-1] “2DG Measurement Protocol” above.