CellCycleAnalysisbyDNAContent(PropidiumIodide).docVIP

Cell Cycle Analysis by DNA Content (Propidium Iodide) Fixation: Wash cells by centrifugation (e.g. 200 x g, 5 min, 4°C) in protein-free buffer, such as Phosphate Buffered Saline without Ca+2 or Mg+2 (PBS). (Optional) Repeat step 1. Resuspend at 2 x 106 cells in 1 ml ICE COLD BUFFER. Cell number will effect staining quality!?Optional: Use pre-coated or silanized polypropylene tubes to minimize sticking. Pre-coat tubes overnight with 2% Bovine Serum Albumin (BSA) in PBS. Vortex gently, slowly adding the cell suspension dropwise to 9 ml of 70% ethanol in a 15 ml polypropylene centrifuge tube (Falcon? Cat. No. [35]2097). ?OR: Vortex gently, slowly adding the cell suspension dropwise to an equal volume of COLD ABSOLUTE ethanol.Optional: Observe cell preparation with a microcope to verify minimum cell clumping. Store at 4°C to - 40°C for AT LEAST 2 hours, 12 - 24 hours is best. Can be stored for up to 2 years before staining. Centrifuge cells at 200 x g, 10 min, 4°C. Resuspend pellet in 3 ml COLD PBS and transfer to Falcon? 12 X 75 mm (Cat. No. [35]2054) polystyrene tubes for staining if other tubes (polypropylene) were used for the fixation steps above. Falcon? Cat. No. [35]2235 have nylon filter caps and will remove clumps. Staining with Propidium Iodide (PI) Wash cells at least once with COLD PBS. Cells may form a diffuse ring-shaped pellet, so centrifuge longer ( e.g. 200 x g, 10 min, 4°C). Resuspend cells in 300 - 500 μl? PI/Triton X-100 staining solution : To 10 ml of 0. 1 % (v/v) Triton X-100 (Sigma) in PBS add 2 mg DNAse-free RNAse A (Sigma) and 0.40 ml of 500 μg/ml PI (e.g., Roche).? Prepare freshly. A stock solution of PI, made by dissolving 1 mg PI? in 2 ml water, can be stored several months at 0° to 4°C. (Or buy 500 μg/ml PI from Roche new Catalog # 11348639001, old Cat. No. 1348639) ? Note: If the RNAse is not DNAse-free, boil a solution of 2 mg RNAse A? in 1 ml water for 5 min. Aliquot and store at -20°C. Incubate 37°C for 15 minutes or for 3