Mastering Cell Counting

Head To Head: Automated Cell Counting vs. Manual Cell Counting

Accurate cell counting is critical to ensure experimental success and reproducibility for cell-based assays and cell culture applications.

Calculating the number of cells is required for monitoring cell proliferation & viability, optimizing cell culture conditions, and preparing cell-based assays.

Cell counting procedures need to be:

Accurate
Reproducible
Cell type independent, i.e., take into consideration cell property variables such as cell clumping, cell shape, and cell populations of varying size

AUTOMATED Cell Counting

Automated cell counters offer an efficient and reliable way to quickly count and analyze a cell population.

Image-Based Counters

Image-based systems use bright field or fluorescent microscopy to capture an image of the cells. Some systems operate on a flow-based imaging methodology where cells are drawn into a capillary and the cells are imaged and counted as they pass a detector. Cell viability can be calculated using dye exclusion methods, such as Trypan Blue. Software analyzes the images based on system-specific parameters such as cell diameter, brightness, and circularity to determine the number of cells and cell viability.

Coulter Counters

Coulter counters measure changes in electrical resistance to determine the number, volume and size of the cells in the sample. Some Coulter counters offer the ability to distinguish live cells from dead cells and cell debris. flow cytometers cell counting

Flow Cytometers

Flow cytometers are not dedicated cell counters and report on relative values, such as the percent of cells in a given sample that have specific properties. The volume of sample counted needs to be determined to calculate the absolute cell count. To accomplish this, samples need to be spiked with fluorescent counting beads as a control.

PROs	CONs
Fast	Initial cost for equipment
Accurate
Reproducable
Eliminates subjectivity
Analysis & graphical display
Data storage & export capabilities
Supports 21 CFR Part 11

MANUAL Cell Counting

Manual cell counting offers an accessible way to determine the concentration of cells in a liquid sample, requiring just a light microscope and hemocytometer.

Cell density (cells/mL)	=	(Average number of cells counted per square) x (Dilution factor)
		Volume of square (mL)

Cell viability can be calculated by treating the cell suspension with cellular dyes, such as Trypan Blue. Live cells do not take up Trypan Blue, dead cells with stain blue.

Cell viability (%)	=	Total viable cells (unstained)
		Total cell (stained + unstained cells)

Hemocytometer

DIMENSIONS	AREA	VOLUME AT 0.1 MM DEPTH
1 x 1 mm	1 mm²	0.0001 mL
0.25 x 0.25 mm	0.0625 mm²	0.00000625 mL
0.20 x 0.20 mm	0.04 mm²	Initial cost for equipment
0.05 x 0.05 mm	0.0025 mm²	0.00000025 mL

1. Apply the cell suspension so that the sample fills the chamber.
Tip: Dilute the cell suspension if necessary. Cells should be uniformly distributed without clumping or overlap on the grid.

2. Using a microscope, count the number of cells in 4 squares.
Tip: The lower the concentration of cells, the more squares should be counted to reduce statistical errors.
Tip: Only count cells that are within the square or on the bottom or left lines; do not count cells that touch the top or right lines of the square.

3. Calculate the density of cells in the suspension.