Cytofix cytoperm plus fixation and permeabilization solution kit

Caspase-9, caspase-8, caspase-3, cleaved-caspase-3, poly(ADP-ribose) polymerase (PARP), XIAP, cIAP-1, cIAP2, Bcl-2, Bcl-xL, and Bax were procured from Cell Signaling Technologies (Beverly, MA, USA) and the GAPDH antibody was procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V-FITC, propidium iodide staining solution, Hoechst33342Solution, BD Cytofix/Cytoperm Plus fixation and permeabilization solution kit(BD (Pharmingen San Jose, CA, USA). The Cell Counting Kit-8 (CCK-8) kit and N-acetyl cysteine (NAC) was obtained from Sigma-Aldrich (St. Louis, MO, United States). z-VAD-FMK was bought from Calbiochem (San Diego, CA, USA). CellROXGreen, MitoSOXRed, andThiolTracker Violet were purchased from Invitrogen (Waltham, MA, USA). Mitopotential kit was purchased from the EMD Millipore Corporation (Danvers, MA, USA).

Leukemic cells after treatment with various doses of embelin were fixed and permeabilized using BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit. 1 x 10⁵ cells were stained with 5 μL H2AX (pS139)-Alexa Fluor 647 antibody and then measured by Flow Cytometry, as described earlier, to quantify the DNA double strand breaks [33 (link)].

Embelin was purchased from Tocris Bioscience (Minneapolis, MN). zVAD-fmk was purchased from Calbiochem (San Diego, CA). Antibodies against caspase-9, caspase-8, Bid, Bcl-xL, phospho AKT and cleaved caspase-3, caspase-3 were purchased from Cell Signaling Technologies (Beverly, MA). Cytochrome c, PARP and GAPDH antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). XIAP antibody was purchased from Abcam (Cambridge, England). Annexin V-FITC, Propidium iodide staining solution, Hoechst 33342 Solution, BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, PE Active Caspase-3 Apoptosis Kit and Apoptosis, BD MitoScreen (JC-1) Kit, DNA Damage and Cell Proliferation Kit were purchased from BD Biosciences (NJ, USA). AKT, XIAP and Scrambled control siRNA were purchased from Qiagen (Valencia, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). 5-(2-Benzothiazolyl)-3-ethyl-2-[2-(methylphenylamino)ethenyl]-1-phenyl-1H-benzimidazolium iodide was purchased from Sigma Aldrich (USA).