Fro coating, 0.2% gelatin works well. Fibronectin works even better. For media, DMEM with pyruvate seems to work well. I would use neonatal mice between day 1 and 4.
Nitrocellulose
Stock solution: 1 cm2 nitrocellulose paper (Sigma Cat No. N8267-5EA). Dissolved in 10 mL Methanol and filtered through a 0.22 µm syringe filter.
Working solution: 1: 10 solution of the stock solution in Methanol.
Nitrocellulose substrate – use 10 µL of the working solution per well in a 24 well plate or 3 µL of the working solution per well in a 96 well plate. If your cells are grown on glass coverslips use 8 µL of the working solution per coverslip. Allow to air dry for five minutes before plating.
These work well too....
Fibronectin (Calbiochem # 341635)coating:
- stock in Millipore (1mg/mL)
- 200uL working solution (100µg/mL Millipore) per well
- incubate 1hr at 37⁰C
- rinse 2 times with sterile Millipore water
Laminin (L2020 from Sigma) coating:
- 500µL of stock Laminin (1mg/mL)
- 49.5mL Culture PBS
- freeze and store in 1mL aliquots @ -20ºC as a 10ug/mL working stock
- 300uL Laminin working stock per well
- incubate overnight in the fridge
- rinse with sterile Millipore water
- let air dry in the cabinet with the UV on for 15 minutes
We find that primary cardiomyocyte isolations from mouse embryos (from day 15 gestation) are much better (beat in culture etc).
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