After the Sindbis virus injection expressing control tdTomato or C99ΔC with tdTomato as bicistronic constructs, the slices were incubated for 24 hr without changing media. At 12 or 18 hours after viral infection, GSI or caspase inhibitors were added to the OTSCs: 10 μM DAPT (565770, EMD Biosciences), 10 μM zVAD (FMK001, pan-caspase inhibitor, R&D Systems), 10 μM zDEVD (FMK004, caspase-3 inhibitor, R&D Systems), 10 μM zYVAD (FMK005, caspase-4 inhibitor, R&D Systems), 10 μM zVEID (FMK006, caspase-6 inhibitor, R&D Systems), and 10 μM zIETD (FMK007, caspase-8 inhibitor, R&D Systems). A drug concentration of 10uM for caspase inhibitors was selected based on the prior determination that was no observable toxicity in dendritic spine density when given to control cultures and this concentration is at or above the Ki of all the inhibitors (data not shown).
Fmk004
Manufactured by R&D Systems
The FMK004 is a laboratory instrument designed for protein detection and quantification. It utilizes a fluorescence-based detection method to measure the concentration of proteins in a sample. The FMK004 is capable of analyzing a wide range of protein samples and provides accurate and reliable results.
Automatically generated - may contain errors
2 protocols using fmk004
1
Inhibition of Caspases and Notch Signaling in Sindbis Virus Infected OTSCs
2
Caspase Inhibition in Differentiated Cells
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After 21 days, differentiated cells were used for caspase inhibition. The cells were seeded at a density of 5000 cells per cm2 and cultured for 6 days (as reported previously4 (link)) in the presence of caspase inhibitors: general caspase inhibitor Z-VAD-FMK and inhibitors for individual apoptotic caspases (Caspase-2,-3,-6,-8,-9). Pharmacological inhibitors (FMK001, FMK003, FMK004, FMK006, FMK007, and FMK008, R&D Systems) were added to the culture medium at a concentration of 100 μM, according to the manufacturer’s instructions and previous studies4 (link). Controls were generated using DMSO as an inhibitor vehicle at the same concentration. The medium with different treatments was changed every 2 days. The effectiveness of the individual caspase inhibitors was verified with Caspase-Glo assays (Supplement 2), and the constant inhibition effect of the FMK inhibitors in cells was confirmed previously38 (link)
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