Methods for counting cells
The invention features methods of quantifying cells in a sample by lysing the cells followed by the measurement of at least one intracellular component. Methods of the invention are especially useful for quantifying small numbers of cells, e.g., over a large surface area or volume compared to the cell size. In a preferred embodiment, methods of the invention are performed using a microfluidic device.
1. A method of counting mammalian cells in a sample, the method comprising:
introducing the sample into a channel, wherein a surface of the channel is coated with binding moieties that bind specifically to a cell surface marker of the mammalian cells;
immobilizing the mammalian cells in the channel by allowing the mammalian cells to bind to the binding moieties;
introducing an isotonic, low conductivity solution into the channel to reduce background conductivity in the channel;
measuring an impedance baseline in the channel;
lysing the cells in the channel by replacing the isotonic, low conductivity solution in the channel with a hypotonic, low conductivity solution; and
measuring an impedance in the channel after cell lysis, a change in impedance from the impedance baseline is indicative of the number of mammalian cells lysed in the channel.
2. The method of claim 1 , wherein measuring the impedance baseline and the impedance in the channel after cell lysis both comprise applying an electric field to the channel at a single frequency between about 100 Hz and about 10 kHz.
3. The method of claim 1 , wherein the change in impedance occurs as a result of a release of intracellular ions from the mammalian cells as a result of the lysing.
4. The method of claim 1 , wherein the change in impedance occurs as a result of a release of nuclei from the mammalian cells as a result of the lysing.
5. The method of claim 1 , wherein measuring the impedance baseline and the impedance in the channel after cell lysis both comprise using an electrode selected from the group consisting of: a top down electrode, an interdigitated (IDT) electrode, and a two rail electrode.
6. The method of claim 1 , wherein the mammalian cells comprise CD4+ lymphocytes and the binding moieties comprise anti-CD4 antibodies.
7. The method of claim 1 , wherein the sample comprises a heterogeneous mixture of cells.
8. The method of claim 7 , wherein the sample is blood.
9. The method of claim 1 , wherein the isotonic, low conductivity solution comprises about 8.5% sucrose and about 0.3% dextrose.
10. The method of claim 1 , wherein the hypotonic, low conductivity solution comprises about 2% sucrose and about 0.07% dextrose.
11. The method of claim 1 , wherein the hypotonic, low conductivity solution comprises a detergent.
12. The method of claim 1 , wherein the channel is a closed microfluidic channel.
13. The method of claim 1 , wherein a change in impedance between the impedance baseline and the impedance in the channel after cell lysis is caused by a change in bulk conductivity of the sample in the channel.
14. The method of claim 1 , wherein an impedance of the isotonic, low-conductivity solution is greater than 1 kΩ.
15. The method of claim 1 , wherein an impedance of the isotonic, low-conductivity solution is greater than 10 kΩ.
16. The method of claim 1 , wherein an impedance of the isotonic, low-conductivity solution is greater than 1 MΩ.