Total Nucleated Cell and Viable Cell Counts
Accurate cell counts are important when conducting all experiments. Ideally both the total nucleated cell (TNC) count and the viability of the cells should be assessed. Researchers generally use Trypan Blue for both TNC and cell viability counts. However for samples containing red blood cells (RBCs), it is often difficult to distinguish nucleated cells from RBCs using Trypan Blue alone.
Total Nucleated Cell Counts
For cell samples that contain RBCs (e.g. whole blood, bone marrow), 3% Acetic Acid with Methylene Blue (Catalog #07060) should be used to perform TNC counts.
The acetic acid lyses red blood cell and white blood cell membranes, leaving white blood cell nuclei stained lightly with methylene blue.
Cell Viability Counts
For samples with possible compromised viability (e.g. frozen samples), one should always determine the viability using Trypan Blue (Catalog #07050).
In the Trypan Blue Exclusion Test, viable cells with intact cell membranes remain unstained, while the non-viable cells with leaky membranes take up the trypan blue dye. Since Trypan Blue is a cytoplasmic stain, acetic acid-lysed cells cannot be used for Trypan Blue staining.
How to Count Cells with a Hemocytometer
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.