Cell cycle test plus dna reagent kit

Cell cycle analysis was performed using Cell cycle test plus DNA reagent kit (BD Biosciences) following the manufacturer’s instructions. Briefly, cells were seeded in 6-well plates at a density of 2×10⁵cells/well. After overnight culture, cells were treated with leptin and/or tamoxifen for the indicated time duration. After washing with PBS and buffer solution, Buffer solution A, solution B, and the propidium iodide solution were sequentially added to the cells, followed by incubation for 15 min in ice on dark. Finally, DNA contents in the cells stained with propidium iodide were determined using a flow cytometer (BD FACSverse). Cell distribution in different phases of the cell cycle was analyzed using BD FACSuite software.

DMSO and Giemsa stain were purchased from Sigma (MO, USA); MTS-PMS assay kit was from Promega (WI, USA); Apoptotic DNA Ladder Kit was from Roche (Basel, Switzerland); JC1 dye was from ThermoFisher Scientific (OR, USA), H₂DCFDA was from Molecular probes (OR, USA); Annexin V-PI assay kit, BDOpt EIA^TMELISA set, Superscript first stand synthesis system was from Invitrogen (CA, USA); Cell Cycle Test Plus DNA reagent kit, 7-AAD and anti-SHP1 antibody were purchased from BD Pharmingen and BD Biosciences (CA, USA); UCP2 antibody was from SantaCruz Biotechnology (CA, USA). All other antibodies were purchased from Cell Signaling Technologies (USA). Mahanine was dissolved in 100% ethanol to make 5000 μM for in vitro studies and dissolved in >5% DMSO for in vivo studies.
Inbred Balb/c mice (20–22 g) were housed in Institute Animal Facilities in CSIR-CDRI and fed a standard diet. All the animal-related experiments were performed in accordance with the National Regulatory Guidelines issued by Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India and used for experiments with prior approval from Institutional Animal Ethical Committee (IAEC)(IAEC approval no: IAEC/2015/42D).