What is Cell Counting

Cell counting is an integral part of determining cell concentrations for plating in culture, determining cell viability, and assessing the results of cell isolation procedures. It is recommended to perform an initial cell count prior to cell isolation; this number can then be compared to the cell count after cell isolation to calculate cell recovery. Additionally, viable cell counts should be performed when a decrease in cell viability may be expected, for example, when working with cryopreserved cells or cells manipulated ex vivo.

We offer Disposable Somatic Cell Count Kit which is composed of Propidium Iodide (PI) for counting somatic cells.

Along with somatic cell counting slides, SCC Kit (CRS-K01 / CRS-K02) is composed of Propidium Iodide (PI) for counting somatic cells.

SCC Kit can be used without diluting raw milk. The measuring range of cell density is 0.05 to 1.15ⅹ106 cells/mL. Each tube has 100 μL reagent of somatic stain solution. Simply add the same volume of the raw milk sample in the tube then every preparing for experiment end. Once the experiment is complete ,the results can be printed through the thermal print. A printed number indicates cell concentration (ⅹ1000/mL) in each channel.

Cell counting can be performed using Trypan Blue or 3% Acetic Acid with Methylene Blue. When performing a total nucleated cell count, 3% Acetic Acid with Methylene Blue is recommended. Acetic acid lyses the cellular membranes, and the methylene blue stains the exposed nuclei. Because mature red blood cells lack nuclei, they are excluded when counting. Alternatively, Trypan Blue is recommended for counting viable mammalian cells. Trypan Blue penetrates the cell membrane, thus it enters the cytoplasm of cells with compromised membranes (dead cells) to stain them blue. The live cells remain intact and can be distinguished from dead cells by their ability to exclude the blue dye. In this case one would count the intact viable cells.

This protocol describes how to perform total nucleated cell counts with 3% Acetic Acid with Methylene Blue, and how to perform viable cell counts by Trypan Blue dye exclusion. Additional cell counting resources and templates to help streamline your assays are also available.

The full grid on a hemocytometer contains nine squares, each of which is 1 mm2. The central counting area of the hemocytometer contains 25 large squares and each large square has 16 smaller squares.

When counting cells that overlap an exterior line or ruling, count only those cells on the top or right-hand line of the large square to avoid counting cells twice. Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed.

To perform the count, determine the magnification needed to recognize the desired cell type and systematically count the cells in selected squares so that the total count is approximately 100 cells, a minimum number of cells needed for a statistically significant count.

For large cells, you can simply count the cells inside the four large corner squares and the middle square. For a dense suspension of small cells, you may wish to count the cells in the four outer and middle squares of the central square or make a more dilute suspension.

Remember if a cell overlaps a line, count it as “in” if it overlaps the top or right-hand line and “out” if it overlaps the bottom or left-hand line.

The area of the middle and each corner square is 1 mm x 1 mm = 1 mm2. The depth of each square is 0.1 mm. Hence, the final volume of each square at that depth is 100 nl.