Quantification of cellular DNA content is a commonly used method for monitoring cell cycle progression. As cells progress through the cell cycle, the amount of DNA ultimately doubles. This doubling can be tracked and used to determine the cell cycle phase (G1, S, and/or G2/M). Cells in the G1 phase have one set of paired chromosomes.

During the S phase cellular DNA begins to double, so that the amount of DNA is between one and two times the amount in G1. Cells in G2/M phase have double the amount of DNA compared to cells in G1 and two sets of paired chromosomes. Thus, the amount of DNA in cells directly reflect in which phase of the cell cycle the cells are.

The Tali® Cell Cycle Kit provides an easy-to-use, optimized, all-in-one solution containing propidium iodide, RNase A, and Triton® X-100 to label cells for cell cycle analysis using the Tali® Image-Based Cytometer.

Materials Required

  • Cells of interest in single cell suspension (appropriate sample concentrations range from 1.105–5.106 cells/mL in the assay)
  • Dulbecco’s Phosphate Buffered Saline (DPBS)
  • Ice cold 70% ethanol in distilled water for cell fixation
  • Tali® Cellular Analysis Slides (Cat. nos. T10794, T10795)

Fix cells

  1. Transfer cells to a conical tube.
  2. Wash cells.
    • Centrifuge at 500 x g for 5 min.
    • Remove medium and resuspend in DPBS.
    • Centrifuge at 500 x g for 5 min.
    • Transfer tubes to ice.
  3. Fix with ice-cold 70% ethanol in dH2O.
    • Remove DPBS.
    • Slowly resuspend cells in ice-cold 70% ethanol to a concentration of 1 x 106 to–5 x 106 cells/mL. To ensure a single-cell suspension of fixed cells, begin the initial addition of 70% ethanol very slowly (dropwise) while gently vortexing. It is very important that cells are fixed into a single cell suspension. Cells can tend to clump during the fixation step. Very slow dropwise addition of the first 1ml of 70% ethanol while gently vortexing will help prevent cells from clumping.
    • Place cells at –20°C overnight. These cells can be kept for weeks at –20°C before staining and analysis.

Stain the cells

  1. Wash cells.
    • Centrifuge cells at 1,000 x g for 5 min at 4°C.
    • Remove ethanol, and resuspend cells in DPBS. Ethanol-fixed cells can be difficult to pellet, and it can be very difficult to see the cell pellet at this step. To reduce the risk of aspirating the cell pellet, it may be advisable to decant the supernatant (instead of aspirating it) after pelleting the cells. If cell density is very low, you can resuspend cells in DPBS + 1% BSA in this step. The addition of BSA will help pellet the cells.
    • Centrifuge cells at 500 x g for 10 min at 4°C.
  2. Stain the cells with the Tali® Cell Cycle Solution
    • Remove DPBS and resuspend the cells in 200 uL of Tali® Cell Cycle Solution to a final cellular concentration of 1 x 105 to–5 x 106 cells/mL (i.e., 2 x 104 – 1x106 cells in 200 ulof Tali® Cell cycle Solution).
    • Incubate at room temperature for 30 min in the dark.
    • Vortex the cells briefly and gently resuspend them before cell cycle analysis using the Tali® Image-Based Cytometer.

Note:  Refer to the protocol for the Tali® Cell Cycle Kit (A10798) for additional details on how to analyze the samples using the Tali® Image-Based Cytometer

Data Analysis

Cell cycle data modeling with the FCS Express™ 4 Image Cytometry software

Download the Application Note