Knowing how many live cells you have and how fast they are dividing is critical for reproducible cell culture and pharmacology experiments.

Hemocytometer Counting

The hemocytometer is a specialized microscope slide with two etched counting chambers, each containing a grid of known dimensions. The grid consists of nine 1 × 1 mm squares, each subdivided into 16 smaller squares. The depth is 0.1 mm, giving a volume of 0.1 µL per 1 mm² square.

1. Mix the cell suspension thoroughly to ensure single cells.
2. Mix 10 µL of cell suspension with 10 µL of 0.4% trypan blue (for viability).
3. Load 10 µL of the mixture into the hemocytometer chamber.
4. Count the cells in the four corner 1 mm² squares (at least 100 cells total for statistical significance).
5. Calculate: cells/mL = (average count per square) × dilution factor × 10⁴.

Trypan Blue Exclusion

Trypan blue is excluded by live cells with intact membranes but enters dead cells, staining them blue. Viability = (live cells / total cells) × 100%. Acceptable viability for most experiments is >90%.

Automated Cell Counters

Automated counters (Countess, Vi-CELL, Cellometer) use trypan blue and digital image analysis to count hundreds of cells in seconds. They reduce operator-to-operator variability and provide size distribution data. Most also calculate viability and can flag cell clumps. Calibrate automated counters against manual hemocytometer counts periodically.

Viability and Cytotoxicity Assays

Beyond trypan blue, several plate-based assays assess viability and cytotoxicity in multi-well formats:

• MTT/MTS assay: mitochondrial dehydrogenases in live cells reduce the tetrazolium dye to a colored formazan (absorbance at 490–570 nm). The signal is proportional to the number of metabolically active cells.
• Resazurin (Alamar Blue): live cells reduce resazurin to fluorescent resorufin. Non-toxic — the same cells can be measured over time.
• LDH release assay: lactate dehydrogenase released from damaged cells into the medium is measured enzymatically (absorbance at 490 nm). Directly measures cytotoxicity.
• ATP assay (CellTiter-Glo): luciferase-based measurement of cellular ATP. Extremely sensitive and linear over a wide dynamic range.

Proliferation Assays

• Growth curve: count cells daily over 5–7 days and plot log cell number vs. time. Doubling time is calculated from the exponential phase.
• BrdU/EdU incorporation: nucleoside analogs incorporated into DNA during S-phase are detected by antibody staining (BrdU) or click chemistry (EdU). Measured by flow cytometry, fluorescence microscopy, or ELISA.
• SRB assay (sulforhodamine B): stains cellular protein. Fixed cells are stained with SRB, and the bound dye is solubilized and measured at 510 nm. Excellent linearity and stability.