Muse cell cycle kit

Human MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS), Penicillin-streptomycin Cocktails and Cystatin C (Catalog number: PHP0044) were obtained from Thermo Scientific (Rockford, IL). SAHA was purchased from Sigma-Aldrich (St. Louis, MO). Muse Cell Cycle kit, Muse Annexin & Dead Cell kit, and Muse Count & Viability kit were from Millipore (Darmstadt, Germany). Human MAPK Antibody Array kit was purchased from R&D Systems (Minneapolis, MN). High Pure RNA Isolation kit and Transcriptor First Strand cDNA Synthesis kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Exprofile Human autophagy Gene qPCR Array kit was obtained from Genecopoeia (Rockville, MD). Power SYBR Green PCR Master mix, RIPA Cell Lysis buffer and BCA Protein Assay kit were from Life Technologies (Austin, TX). Polyclonal anti-beclin-1 antibody, polyclonal anti-Atg3 antibody, polyclonal anti-Atg5 antibody, polyclonal anti-Atg7 antibody, polyclonal anti-Atg12 antibody, polyclonal anti-Atg16 antibody, polyclonal anti-Atg4A antibody, polyclonal anti-Atg4B antibody, polyclonal anti-Atg9B antibody, polyclonal anti-LC3II antibody were obtained from Abcam Inc (Cambridge, MA). Protease inhibitor and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cell cycle distributions of untreated and chromene C1- and C2-treated MDA-MB-231 and Hs578T cells were analyzed using a Muse™ Cell Cycle Kit (Millipore), with all procedures following the instructions provided by the manufacturer. Cells grown in 6 cm dishes were treated with or without C1 and C2 for 24 h. Cell cycle analysis was then performed as previously described [27 (link)], using the Muse™ Cell Analyzer. All data collected were analyzed using FlowJo version 7.6.5.

The distribution of cell population in each phage of cell cycle was determined using a Muse™ Cell Cycle Kit (Millipore Co.), according to the manufacturer’s instructions. Briefly, NHDF cells were treated with 55 mJ UV, and subsequently with MED or CPO for 24 h. After centrifugation, the harvested cells were fixed with 70% EtOH for 3 h at −20 °C and incubated with the 200 μL of Cell Cycle Reagent at 37 °C for 30 min. The number of cells in each phase was analyzed by a Muse^TM Cell Analyzer (Millipore Co.).

Cell cycle analysis was performed with Muse™ Cell Cycle kit (Milipore Corp., MA, USA) according to the manufacturer’s protocol. The cell cycle distribution was determined using the flow cytometry-based system Muse™ Cell Analyzer (Merck, Germany).

Aβ-amyloid peptides (China Peptides, China) were prepared as described previously [17 (link)]. NSC harvest and routine culture was as described previously [18 (link),19 (link)]. For the neural colony-forming assay, cells were seeded in a semi-solid gel matrix made with a 2:1 solution of proliferation medium and collagen. After day 21, neurospheres were counted and their diameter measured using NIS-Elements (Nikon Adelaide, Australia) software. Cell cycle analysis was performed using the Muse Cell Cycle Kit (Millipore, Bayswater, Victoria AUS). For plate and blotting assays cells were cultured as an adherent monolayer on a 1:1 poly-D-lysine-laminin matrix. Cellular ATP content was measured using Life Sciences’ ATP assay (Invitrogen, Mulgrave, Victoria AUS). Immunodetection methods have been described previously [18 (link)-20 (link)]. Expanded methodology is provided in Additional file 1.

The cell cycle distribution analysis in DMSO- or carnosol-treated MDA-MB-231 cells was performed with the Muse Cell Analyzer (Millipore, Hayward, CA, USA) using the Muse cell cycle kit (Millipore, Hayward, CA, USA) according to the manufacturer's instructions. Briefly, cells grown onto 6 cm culture dish were treated with DMSO (vehicle) or various concentrations of carnosol. After 24 h of treatment, cells were collected by trypsinization, washed in PBS and resuspended in complete media and the Muse cell cycle test reagent was then added to each test tube. Cells were then incubated for 30 min at room temperature in the dark. After staining, the cells were processed for cell cycle analysis. Percentage of cells in G0/G1, S and G2/M phases were determined using the FlowJo software.