- Luciferase assay substrate (Promega, E1500).
- Prepare the luciferase assay reagent: Add 10 ml of luciferase
assay buffer to the vial containing the lyophilized luciferase
assay substrate (seeNote 6). - Opaque white 96-well plate.
- PerkinElmer 2030 program.
- Plate-reading luminometer (Victor X4, PerkinElmer).
2.6 Evaluation ERK1/
2 Signaling Pathway
Except for antibodies, see Subheading 2.3 for the materials
required.
Antibodies- Anti-ERK1/2 (Cell signaling, 9102): for IB 1:1000 in TBS-T,
5% (w/v) BSA. - Anti-ERK1/2 Thr202/Tyr204 (Cell signaling, 9101): for IB
1:1000 in TBS-T, 5% (w/v) BSA. - Anti-eEF2 (Thermo scientific, PA5–17794): for IB 1:1000 in
TBS-T, 5% (w/v) BSA. - Goat anti-rabbit IgG HRP-conjugated (Sigma, A0545): for IB
1:20,000 in TBS-T, 5% (w/v) BSA.
3 Methods
3.1 Isolation
and Culture
of Neonatal Rat
Ventricular
Cardiomyocytes
for Protein Synthesis
and Signaling Analysis
Carry out all procedures in sterile conditions under the hood except
heart harvesting.
Trypsin digestion of neonatal hearts- Sacrifice rat pups (typically 60 pups are used) by cutting the
head under aseptic conditions. Remove and place the hearts in
a petri dish containing 30 ml of HBSS. - Under the hood, transfer all hearts in a second petri dish
containing 30 ml of HBSS, and cut them in four pieces with a
scalpel. - With a 25 ml pipette, transfer the 30 ml of HBSS containing
the pieces of heart into 50 ml flask containing 30 mg of trypsin
in order to make a trypsin solution of 1 g/L. - Digest the hearts at 4C in a stirring plate for 3–4 h.
Collagenase type II digestion - At the end of trypsin digestion, add 20 ml of warm full IMDM
medium, and place the flask in a stirring water bath at 37C for
5 min to stop the reaction. - Discard the supernatant as much as you can, and leave the
cardiomyocyte pieces in the bottom.
328 Florence Mailleux et al.