Cell Counting

The TC20™ automated cell counter has two counting modes. Depending on the sample type, the user-defined gates can be either enabled or disabled.

Samples with Multiple Cell Populations (stem cells and other primary cells)

User-defined gates must be enabled. Once the user-defined gates are enabled, a histogram will be displayed at the beginning of each count and the user can select the population of interest by adjusting the position of the cell size gates. Only objects within the designated range will be analyzed as cells; those with diameters outside of the size range will be excluded from the cell count.

If the user-defined gates are disabled, the cell counting algorithm will automatically determine the population to count without any user input. The algorithm will count the cells of the most predominate cell type that typically comprise the largest peak, which may not be the population the user wants to count.

Gating setup window.		Cell size gating window.

To make counting of such samples easier, when counting multiple sample replicates, the TC20 cell counter can save the positions of gates and apply them to subsequent counts (choose Yes in the “Use saved gates” field in Gating setup).

If the Image preview is On, an image of cells will be displayed during gating. In the image, only cells with diameters between the selected size gates will be marked with a yellow dot.

Immortal Cell Lines

When counting immortal cell lines or other samples that are composed of cells with similar cell size, the user-defined gates can be disabled. The cell counting algorithm then automatically identifies the cell population of interest without requiring user input.

The data below show the TC20™ automated cell counter is compatible with a broad range of cell types, such as cell lines, primary cells (from tissue or blood), and stem cells. The TC20 cell counter can count cells with a 6–50 µm cell diameter and within a broad concentration range of 5 x 10⁴–1 x 10⁷ cells/ml, which eliminates the need to dilute cells, thus reducing the error associated with sample dilutions prior to counting.

The TC20 automated cell counter demonstrates accurate cell counts across a range of cell sizes. Small (PBMC, Jurkat), medium (HeLa), and large (MEF) cells were counted with a hemocytometer, a TC20 automated cell counter, and a competitor's image-based automated cell counter. The TC20 counter and hemocytometer cell counts showed no statistically significant differences. Precision is indicated by the standard deviations; error bars represent average standard deviations. Cell counts on the TC20 counter were performed on one instrument with four sample replicates.

The TC20 automated cell counter demonstrates accurate counts of viable cells. Pan T cells mixed with trypan blue (1:1) were counted with a hemocytometer, a TC20 automated cell counter, and a competitor’s image-based automated cell counter. The TC20 counter and hemocytometer cell counts showed no statistically significant differences. Precision is indicated by the standard deviations; error bars represent average standard deviations. Cell counts on the TC20 counter were performed on four different instruments with five sample replicates.

The TC20™ automated cell counter uses microscopy with auto-focus that analyzes multiple focal planes to identify the best plane. The cell counting algorithm then uses the image acquired from the best focal plane to identify cells and exclude debris, thereby calculating the total cell count.

Using auto-focus instead of subjective manual focusing is especially important when assessing cell viability because an incorrectly selected focal plane will lead to inaccurate results.

If trypan blue is detected along with the total cell count, the TC20 counter assesses cell viability. The conventional method of analyzing viability using a single focal plane can lead to inaccurate conclusions because light scattering and the alignment of cells at different heights in a counting chamber can change the appearance of cells — live cells may appear to be dead and vice versa. To determine if cells are viable, the TC20 counter analyzes each cell using images acquired from multiple focal planes during the focusing step.