A scalable plate reader-based protocol to measure mitochondrial respiration in embryonic hearts

Figures

• 

  Figure 1. Data reduction setup for lifetime Two data sets are input to the transformation and the formula is applied to all wells.

• 

  Figure 2. Kinetic analysis setup for OCR Lifetime is input to the kinetic analysis and “Max V” is the calculation type.

• 

  Figure 3. Calculation options for kinetic analysis of OCR All available reads are used and RFU per minute is selected for the units.

• 

  Figure 4. Plate Layout for signal to blank optimization. “SC” is signal control and “BLK” is blank

• 

  Figure 5. Representative basal signal trace from one wild-type E14.5 heart (N = 1 embryonic heart).

• 

  Figure 6. Representative signal trace after 10 μM FCCP exposure from one wild-type E14.5 heart (N = 1 embryonic heart).

• 

  Figure 7. Representative signal traces after 5 mM KCN/30 μM rotenone exposure from one wild-type E14.5 heart (N = 1 embryonic heart).

• 

  Figure 8. Quantification of oxygen consumption rates after data reduction and rate conversion Plot shows basal, uncoupled and KCN-inhibited rates (N = 3-4 embryonic heart per condition). Error bars represent mean SEM; Statistical significance was determined using a one-way ANOVA for comparisons involving three or more groups and assuming normal distribution of the data. ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

• 

  Figure 9. Representative lifetime signal trace with a negative slope (N = 1 embryonic heart).

• 

  Figure 10. Representative lifetime signal trace with a delayed upward trend (N = 1 embryonic heart).

• 

  Figure 11. Representative lifetime signal trace with a brief upward trend (N = 1 embryonic heart).

• 

  Figure 12. Representative traces showing insufficient inhibition by KCN and rotenone (N = 6 embryonic hearts).