Quantitation of cellular DNA and cell numbers using element labeling
View Patent ↗Methods and kits for the quantitation of cellular DNA and cell numbers are provided. Passive element uptake, element-labeled DNA intercalators, and element labeled affinity reagents are used to quantify DNA and cells. The DNA and the cells are analyzed by elemental analysis, including ICP-MS. The methods and kits provide a fast and accurate analysis of cellular DNA and cell numbers.
1. A method for determining the phase of a cell cycle in a sample of cells, comprising:
(a) determining the number of cells in the sample;
(b) fixing and permeabilizing the cells;
(c) exposing the sample of cells to an element containing intercalator under conditions appropriate to incorporate the intercalator into the DNA within the cells of the sample;
(d) removing unbound intercalator;
(e) measuring the content of the labeled DNA within the cells by elemental analysis of the element;
(f) determining the amount of DNA per cell; and
(g) determining the phase of the cell cycle from the amount of DNA per cell.
2. The method of claim 1 comprising an additional step prior to step (a) of incubating the cells with cell cycle perturbing agents to arrest the cells in a particular phase of the cell cycle.
3. The method of claim 2 wherein the cell cycle perturbing agents are selected from the group consisting of thymidine and nocodazole.
4. The method of claim 1 where after step (d) the cells are lysed and the lysate is analyzed for the intercalator.
5. The method of claim 1 wherein step (e) is conducted using a mass spectrometer-based flow cytometer.
6. A method for determining the phase of a cell cycle in a sample of cells, comprising:
(a) fixing and permeabilizing the cells;
(b) exposing the sample of cells to an element containing intercalator under conditions appropriate to incorporate the intercalator into the DNA within the cells of the sample;
(c) removing unbound intercalator;
(d) measuring the content of the labeled DNA within the cells by using a mass spectrometer-based flow cytometer;
(e) determining the amount of DNA per cell; and
(f) determining the phase of the cell cycle from the amount of DNA per cell.
7. The method of claim 6 comprising an additional step prior to step (a) of incubating the cells with cell cycle perturbing agents to arrest the cells in a particular phase of the cell cycle.
8. The method of claim 7 wherein the cell cycle perturbing agents are selected from the group consisting of thymidine and nocodazole.
9. The method of claim 6 where after step (c) the cells are lysed and the lysate is analyzed for the intercalator.