) Evaluation of attainable interferences of Red1-C1 fluorescence with FRET ?Further

De Familia Escosteguy
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Molecular Neurodegeneration (2016) 11:Page 6 ofTable 1 List of experiments, their rationale and most AM152 biological activity important outcomes (Continued)12 Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1-Surv ?Medium 1 (horse serum) ?4 DIV ransfection ?Medium title= jasp.12117 1 (horse serum) ?two DIV ?FRET on fixed OCCs None ?Evaluation of the effects of survivin overexpression on basal levels of caspase three activation ?Evaluation with the impact of survivin on cell survival ?Survivin reduces basal caspase three activity ?Survivin promotes cell survival None ?Process control: a) Evaluation of co-transfection efficiency; b) Evaluation of attainable interferences of Red1C1 fluorescence with FRET ?Additional morphological identification of transfected cells ?Exp. #14 handle ?a) Co-transfection rate is one hundred  ; b) title= jir.2012.0117 Red1-C1 doesn't interfere with FRET ?Confirmation of Exp #4: caspase three is constitutively active in CGCs ?Transfected cells are for the most CGCs 14 Double pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-C1-Surv ?Medium 1 (horse serum) ?four DIV ?Transfection ?Medium two (Neurobasal) ?two DIV ?FRET on fixed OCCs ?Medium 1 (horse serum) ?four DIV ?Transfection ?Medium 2 (Neurobasal) ?1 DIV ?FRET on alive OCCs None ?Evaluation in the effects of survivin overexpression on basal levels of caspase 3 activation ?Evaluation in the effect of survivin on cell survival ?Survivin reduces caspase 3 activity ?Survivin promotes cell survival ?25 mM KCl ?1 mM NMDA ?1 mM KA ?100?00 M A23187 ?50 M?5 mM H2O2 ?60 mM KCl ?one hundred mM H2O2 ?Measurement of caspase 3 activity just after induction of apoptosis ?A subpopulation of CGCs is insensitive to apoptosis induction and does not display modifications in ECFPem/Venusem ?Other cells display indicators of sufferance ?Real time monitoring of caspase three in basal circumstances ?Measurement of caspase 3 activity following depolarization or oxidative pressure ?Caspase 3 activity may be measured in actual time experiments ?Enhance of ECFPem/Venusem following K+ depolarization but not cell death ?Only tendency to boost of ECFPem/Venusem following oxidative strain. Reduction within the variety of transfected cellsDouble pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-CSingle pcDNA-SCAT3 DEVG (handle probe) or pcDNA-SCAT3 DEVD (FRET probe)Single pcDNA-SCAT3 DEVD (FRET probe)from 0.55 ?0.06 (pre-bleach) to 1.10 ?0.10 (post-bleach) (n. cells =12, t-Test: P = 0.0001). Remarkably, it was calculated that the ECFPem/Venusem normalized value changed from 0.43 to 1.11 in HeLa cells transfected with pSCAT3-DEVD and challenged with tumor necrosis factor -cycloheximide, a strong activator of Casp3 [24].) Evaluation of possible interferences of Red1-C1 fluorescence with FRET ?Additional morphological identification of transfected cells ?Exp #12 handle ?a) Co-transfection rate is 100  ; b) Red1-C1 will not interfere with FRET ?Confirmation of Exp #3: caspase 3 is constitutively active in CGCs ?Transfected cells are for essentially the most CGCs?Medium 1 (horse serum) ?four DIV ?Transfection ?Medium two (Neurobasal) ?2 DIV ?FRET on fixed OCCsSingle pcDNA-SCAT3 DEVG (FRET manage probe)?Medium 1 (horse serum) ?4 DIV ?Transfection ?Medium 1 (horse serum) ?2 DIV ?FRET on fixed OCCs?Medium 1 (horse serum) ?four DIV ?Transfection ?Medium 1 (horse serum) ?two DIV ?FRET on fixed OCCsSingle pcDNA-SCAT3 DEVG (FRET handle probe)Single pcDNA-SCAT3 DEVD (FRET probe)Ac-DEVD-CMK 100 M?Medium 1 (horse serum) ?four DIV ?Transfection ?Medium 2 (Neurobasal) ?two DIV ?FRET on fixed OCCsDouble pcDNA-SCAT3 DEVD (FRET probe) + pHcRed1-CLossi et al.