Caspase-3 and PARP Cleavage Assay

After 48 h of treatment with NSP‐B at varying lower doses (0.1, 0.5, 1, 2.5, and 5 µM), cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus fixation and permeabilization solution kit. Anti‐active Caspase‐3‐BV605 and PARP Cleaved Form‐AF700 antibodies were used to stain 0.3 × 10⁶ cells in HBS Hank's balanced salt solution (HBSS) for 30 min. The cells were resuspended in the same solution after being rinsed once more with HBS. Flow cytometry using a BD LSR Fortessa analyzer was used to determine the percentage of cells with activated caspase‐3 and cleaved PARP (Iskandarani et al., 2016 (link)).

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Kuttikrishnan S., Ahmad F., Mateo J.M., Prabhu K.S., El‐Elimat T., Oberlies N.H., Pearce C.J., Akil A.S., Bhat A.A., Alali F.Q, & Uddin S. (2023). Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway. Cell Biology International, 48(2), 190-200.

Publication 2023

BD Cytofix/Cytoperm plus fixation and permeabilization solution kit BD LSR Fortessa analyzer

Corresponding Organization :

Other organizations : Hamad Medical Corporation, Qatar University, Jordan University of Science and Technology, University of North Carolina at Greensboro, Mycosynthetix (United States), Qatar Airways (Qatar)

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Variable analysis

independent variables

NSP-B concentration (0.1, 0.5, 1, 2.5, and 5 μM)

dependent variables

Percentage of cells with activated caspase-3
Percentage of cells with cleaved PARP

control variables

Treatment duration (48 h)
Cell fixation and permeabilization using BD Cytofix/Cytoperm plus fixation and permeabilization solution kit
Antibody staining (anti-active Caspase-3-BV605 and PARP Cleaved Form-AF700) for 30 min
Cell resuspension in HBS (Hank's balanced salt solution)
Flow cytometry using BD LSR Fortessa analyzer

controls

No positive or negative controls were explicitly mentioned in the provided information.

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