小中大下面的是我们这的前辈写出来的protocol，我们一直也用的是这个，仅供参考！
Preparation:
0.1% gelatin: 0.1g gelatin in 100ml PBS. After high pressure sterilization, gelatin dissolve and solution will be clear. Store at 4 ℃.
Digest solution: 0.07% Trypsin with 0.05% Collagenase TypeⅡin HBSS.
Coating methods: Add 50ul 0.1% gelatin to every well. Move the 96-well plate in 4°C. Overnight. Then get rid of gelatin solution. Move the plate to normal incubator.
Methods:
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996).
Cardiac ventricular myocytes were prepared from 1- to 2-day-old Sprague-Dawley rats and rats would be kept in warm temperature.
Cutting and isolation of hearts:
Rats are sterilized by 75% alcohol. Cut chest wall and isolate heart by scissors. Hearts from neonatal rats should be rapidly excised, rinsed in ice cold DMEM, washed to remove blood and debris. Remove atriums from isolated hearts. Ventricles are left and moved in a bottle which with HBSS. Then ventricles are shredded into tissue fragments (1mm3). Get rid of HBSS. Add digest solution, move into normal incubator to begin digestion.
Myocytes digestion:
Tissue fragments are digested by stepwise trypsin and collagenaseⅡ. Each step needs 8 min, shake bottle every 4 min. Use glass pipette to blow the fragments. Aspirate supernatant and move into another tube with 10% FBS DMEM to stop digestion. Repeat the steps (4-5 step) until the fragments disappear. Centrifuge cell suspension with 800 rpm for 5min. And get rid of the suspension. Now the cells are in the bottom of tube. Add DMEM (10%FBS) and re-suspend the cells. Filter cells with filter (200 mesh).
Myocytes enrichment:
The dissociated cells are preplated to 25cm2 culture flask and back to incubator. Use 60 min differential adhesion to enrich the culture with myocytes. The nonadherent myocytes suspension is moved to a tube. The cells are counted and adjusted to a desirable concentration by adding DMEM with 10%FBS.
Myocytes plating:
Add 100ul cell suspension to each well. Cells are cultured in 96-well plates and are maintained at 37°C in a humidified incubator with 5% CO2.Change the medium at 48 hour. And put the plate in the station and record the impedance signaling. After 12h, cells would be used for experiments.
