Table 1 List of experiments, their rationale and main results
Exp # | Culture conditions | Transfection protocol | Treatment(s) | Experiment rationale |
|---|---|---|---|---|
Main results | ||||
1 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 1 (horse serum) – 2 DIV • FRET on fixed OCCs | Single pcDNA-SCAT3 DEVG (FRET control probe) | None | • Assessment of theoretical probe efficiency by acceptor photobleaching • Exp #2 control: confirmation that cellular caspase 3 cannot cleave the mutated DEVG sequence |
• FRETeff can be measured in OCCs | ||||
2 | Single pcDNA-SCAT3 DEVD (FRET probe) | None | • Calculation of ECFPem/Venusem in serum-containing medium to measure basal caspase 3 activation | |
• Caspase 3 is constitutively active in CGCs • Presence of serum reduces basal ECFPem/Venusem (means that caspase 3 activity is reduced) | ||||
3 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 2 (Neurobasal) – 2 DIV • FRET on fixed OCCs | Single pcDNA-SCAT3 DEVG (FRET control probe) | None | • Assessment of theoretical probe efficiency by acceptor photobleaching • Exp #4 control: confirmation that cellular caspase 3 cannot cleave the mutated DEVG sequence |
• FRETeff can be measured in OCCs | ||||
4 | Single pcDNA-SCAT3 DEVD (FRET probe) | None | • Assessment of ECFPem/Venusem in serum-free medium to measure basal caspase 3 activation | |
• Caspase 3 is constitutively active in CGCs | ||||
5 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 1 (horse serum) – 2 DIV • FRET on fixed OCCs | Double pcDNA-SCAT3 DEVG (FRET control probe) + Casp3 shRNA control clone | None | • Negative control for caspase 3 RNAi |
• ECFPem/Venusem after transfection with FRET control probe is not modified by co-transfected control shRNA clone | ||||
6 | Multiple pcDNA-SCAT3 DEVG (FRET control probe) + Casp3 shRNA clones 1–4 | None | • Negative control for caspase 3 RNAi | |
• ECFPem/Venusem after transfection with FRET control probe is not modified by co-transfected Casp3 shRNA clones | ||||
7 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 1 (horse serum) – 2 DIV • FRET on fixed OCCs | Double pcDNA-SCAT3 DEVD (FRET probe) + Casp3 shRNA control clone | None | • Negative control for caspase 3 RNAi |
• ECFPem/Venusem after transfection with FRET probe is not modified by co-transfected control shRNA clone | ||||
8 | Multiple pcDNA-SCAT3 DEVD (FRET probe) + Casp3 shRNA clones 1–4 | None | • Demonstration of probe specificity by caspase 3 RNAi | |
• ECFPem/Venusem after transfection with FRET probe is reduced by co-transfected Casp3 shRNA clones • Constitutively active caspase 3 can be effectively silenced by RNAi | ||||
9 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 1 (horse serum) – 2 DIV • FRET on fixed OCCs | Single pcDNA-SCAT3 DEVG (FRET control probe) | Ac-DEVD-CMK 100 μM | • Negative control for caspase 3 inhibition |
• ECFPem/Venusem after transfection with FRET control probe is not modified in the presence of caspase 3 inhibitor | ||||
10 | Single pcDNA-SCAT3 DEVD (FRET probe) | Ac-DEVD-CMK 100 μM | • Demonstration of probe specificity by specific inhibition of caspase 3 | |
• ECFPem/Venusem after transfection with FRET probe is reduced by caspase 3 inhibitor • Constitutively active caspase 3 can be effectively inhibited by Ac-DEVD-CMK | ||||
11 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 2 (Neurobasal) – 2 DIV • FRET on fixed OCCs | Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1 | None | • Method control: a) Evaluation of co-transfection efficiency; b) Evaluation of possible interferences of Red1-C1 fluorescence with FRET • Further morphological identification of transfected cells • Exp #12 control |
• a) Co-transfection rate is 100 %; b) Red1-C1 does not interfere with FRET • Confirmation of Exp #3: caspase 3 is constitutively active in CGCs • Transfected cells are for the most CGCs | ||||
12 | Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1-Surv | None | • Evaluation of the effects of survivin overexpression on basal levels of caspase 3 activation • Evaluation of the effect of survivin on cell survival | |
• Survivin reduces basal caspase 3 activity • Survivin promotes cell survival | ||||
13 | • Medium 1 (horse serum) – 4 DIV •Transfection • Medium 1 (horse serum) – 2 DIV • FRET on fixed OCCs | Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1 | None | • Method control: a) Evaluation of co-transfection efficiency; b) Evaluation of possible interferences of Red1-C1 fluorescence with FRET • Further morphological identification of transfected cells • Exp. #14 control |
• a) Co-transfection rate is 100 %; b) Red1-C1 does not interfere with FRET • Confirmation of Exp #4: caspase 3 is constitutively active in CGCs • Transfected cells are for the most CGCs | ||||
14 | Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1-Surv | None | • Evaluation of the effects of survivin overexpression on basal levels of caspase 3 activation • Evaluation of the effect of survivin on cell survival | |
• Survivin reduces caspase 3 activity • Survivin promotes cell survival | ||||
15 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 2 (Neurobasal) – 2 DIV • FRET on fixed OCCs | Single pcDNA-SCAT3 DEVG (control probe) or pcDNA-SCAT3 DEVD (FRET probe) | • 25 mM KCl • 1 mM NMDA • 1 mM KA • 100–200 μM A23187 • 50 μM–25 mM H2O2 | • Measurement of caspase 3 activity after induction of apoptosis |
• A subpopulation of CGCs is insensitive to apoptosis induction and does not display changes in ECFPem/Venusem • Other cells display signs of sufferance | ||||
16 | • Medium 1 (horse serum) – 4 DIV • Transfection • Medium 2 (Neurobasal) – 1 DIV • FRET on alive OCCs | Single pcDNA-SCAT3 DEVD (FRET probe) | • 60 mM KCl • 100 mM H2O2 | • Real time monitoring of caspase 3 in basal conditions • Measurement of caspase 3 activity after depolarization or oxidative stress |
• Caspase 3 activity can be measured in real time experiments • Increase of ECFPem/Venusem after K+ depolarization but not cell death • Only tendency to increase of ECFPem/Venusem after oxidative stress. Reduction in the number of transfected cells |