To further characterize N2ICD-mediated apoptosis, we first used a PhiPhiLux™ system to detect real-time activation of caspase in live cells.
A remarkable experiment demonstrated that while contact between the male tarsus and a female's abdomen provokes real-time activation of P1 neurons, the presence of cVA attenuates this response [ 53 ], demonstrating a role for P1 neurons in integrating gustatory and olfactory information.
To monitor the activation stage of signaling molecules in real time, including the activation of Rho-family GTPases, fluorescence resonance energy transfer (FRET) is a valuable technique.
By transplanting these cells into HLA matched recipients we were able to image in real time NF-κB activation in knee joints of mice with IL-1 or SCW-induced inflammation using a cooled-charge-coupled device (CCCD) camera.
Note that, in the condition without the online cursor feedback (experiment 2), the timing of the reaching end was calculated by this method in real time and the cursor activation coincided with this timing.
Overall, although carried out as independent experimental systems, our results show that correlating the two critical events of apoptosis, cyt. c release and caspase activation as analyzed in real time, can differentiate direct caspase activation from classical apoptosis where cyt. c release remains as an initiator for caspase activity.
Thereby, we could achieve a local bidimensional surface receptors engagement in well-defined molecular and mechanical conditions and carry out real time imaging of the activation processes through visualization of [Ca2+]i and actin polymerization.
The mRNA expression was determined by real time PCR, and protein activation was measured by western blotting.
The thermal profile for quantitative real time PCR was: initial activation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s.
Closer analysis of the results of cyt. c release and caspase activation in real time by employing these cell-based tools indicate that cyt. c release preceded caspase activation upon small molecule PAC-1 treatment similar to other drugs.
To further characerize the macrophage activation state, real time gene expression analysis on the ATM/ATL fraction of the epididymal adipose was performed.
