Measurement of the labile iron pool

Reagents

• Calcein-AM (Invitrogen: C1430)
• Pyridoxal isonicotinoyl hydrazine (PIH; Abcam: ab145871)
• MEM (glutamine- and phenol red-free, Gibco: 51200038) supplemented with 1x MEM non-essential amino acids solution (Gibco: 11140035), 100 µg/ml bovine serum albumin, insulin (10 mg/l)-transferrin (5.5 mg/l)-sodium selenite (6.7 μg/l) media supplement (ITS; Gibco: 41400045), 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma-Aldrich: G6784).

Method

1. 2000 cardiomyocytes are seeded in 96 well plate (1 well = 200 μl).
2. Thirty min after seeding the cells, aspirate 150 μL medium, and then add 150 μL medium containing with or without 10 μM ferrostatin-1 (Sigma Aldrich: SML0583) for 30 min.
3. Thirty min later, aspirate 150 μL medium, and then add 150 μL medium with 10 or 20 μM erastin (Sigma Aldrich: E7781), 10 or 100 μM isoproterenol (Sigma Aldrich: I5627).
4. After stimulation, the cells are washed by medium three times (discard 150 μl medium) and incubated with 1 μM calcein-AM in 150 μL medium at 37°C for 10 minutes.
5. The cells are washed three times with the medium (discard 150 μl medium).
6. The fluorescence is measured using a fluorescence microplate reader.
7. Then, the cells are treated with 10 μM pyridoxal isonicotinoyl hydrazine (PIH; Abcam: ab145871) at 37°C for 10 minutes.
8. The cells are washed three times with the medium (discard 150 μl medium).
9. The fluorescence is measured again in a fluorescence microplate reader.
10. The changes in fluorescence (DF) upon PIH treatment is calculated for each sample.

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