Table 1.
Comparison of different methods used to assess metabolic activity and cell proliferation
| Methodology | Advantages | Disadvantages | Technical notes | Can it quantify cell numbers? | |
|---|---|---|---|---|---|
| Haemocytometer | - Low cost | - Need to obtain single cell suspension | - Use in combination with viability dye to differentiate viable from dead cells | Yes | |
| - Direct counting | - Operator variability | - Vortex sample and recount if cell clumps appear | |||
| - No elaborate equipment required | - High error | - Beware of double counting within the grid lines | |||
| - Easy to carry out | - Use larger sample size to reduce error | ||||
| NucleoCounter | - Quick | - High cost $4 per cassette/per run | - Cell penetration and stabilizing solution is needed | Yes | |
| - Easy to carry out | - Cassettes are light sensitive | ||||
| - No operator variability | |||||
| [3H] thymidine | -Strong linear correlation with increasing cell numbers | - High cost | - After labelling strong lysis buffer is needed to permeabilize and disaggregate the culture to obtain homogenous cell suspension | No | |
| - Maintenance of equipment/ infrastructure for radioactivity work | |||||
| - Health risks associated with radioactivity | |||||
| - Need to obtain homogenous suspension | |||||
| - Indirect measurement | |||||
| - Accuracy in high cell density cultures is depended on diffusion efficiency of [3H] thymidine to reach all cells | |||||
| Flow cytometry | - High accuracy | - No reuse of cells, therefore indirect cell count | - Big technical challenge to create a suspension from single cells without any clumping | No | |
| - High reproducibility | - Time consuming with low cell numbers | - Cells cannot be reused unless using FACS- high risk of contamination | |||
| - Inexpensive if flow cytometer is available | |||||
| - No extra staining technique necessary | |||||
| MTT/MTS | - Cost: 37 c/test | - Variable metabolic behaviour under different cell culture conditions | - End-point assay | No | |
| - Easy to carry out | - Indirect measurement | - Converted formazan has to be released from the cells in order to measure the absorbance of the dye | |||
| - Complete kits are available | - No standard provided | - Plan proper controls and reagent blanks | |||
| - Phenol red free media required due to interference with assay dyes | - Establish your own standard curve for each cell line to be used | ||||
| - Dye is not water soluble thus volatile organic solvent is needed | - Use in combination with live/dead staining to confirm the results | ||||
| WST-1 | - Cost: 30 c/test | - Variable metabolic behaviour under different cell culture conditions | - Protect from light | No | |
| - Water soluble | - Indirect measurement | - Plan proper controls and reagent blanks | |||
| - One step easy to apply assay | - No standard provided | - Establish your own standard curve for each cell line to be used | |||
| - Phenol red free media required due to interference with assay dyes | - Use in combination with live/dead staining to confirm the results | ||||
| Hoechst dye | - Sensitive to DNA conformation and chromatin state in cells | - Detection of higher cell numbers from 1000 on | - Protect samples from light | No | |
| - Easy to carry out | - Low fluorescence gradient | - End-point assay | |||
| - Cell permeable stain | - AT-selectivity | - Establish your own standard curve for each cell line to be used | |||
| - Health risk for user | - Special disposal regulations | ||||
| - Mutagenic potential | |||||
| AlamarBlue | - Low cost 10 c/assay | - Variable metabolic behaviour under different cell culture conditions | - Protect samples from light | No | |
| - Non-toxic | - Indirect measurement | - Carry out trials before the actual run to determine optimal reagent concentration and incubation time to prevent signal saturation | |||
| - Easy to carry out | - No standard available from supplier, needs to be performed by user for every cell line to be tested | - Interaction with serum concentration and pH indicator phenol red | |||
| - Plan proper controls and reagent blanks with each run to eliminate data ambiguities | |||||
| PicoGreen | - Cost: 33 c/test | - Several compounds effect the signal intensity in a linear behaviour | - Protect samples from light | Approximate | |
| - ds-DNA specific | - Black plates are required | - Plan proper controls and reagent blanks with each run to eliminate data ambiguities | |||
| - Direct correlation with proliferation | - Subject to signal quenching | - Cell proliferation measured in DNA amount can be compared across specimen groups | |||
| - DNA standard provided | - DNA must be released by freeze thaw cycles or treatment with lysis reagent | - Accurate DNA amounts can be measured provided a representative DNA suspension is obtained | |||
| - Very sensitive | |||||
| - Highly reproducible | |||||
| CyQuant | - Cost: 42 c/test | - Detection of nucleic acids DNA and RNA | - Protect samples from light | Approximate | |
| - Direct correlation with proliferation | - RNAse treatment required for detection of DNA and vice versa | - Plan proper controls and reagent blanks with each run to eliminate data ambiguities | |||
| - DNA standard provided | - Black plates are compulsory | - Cell proliferation measured in DNA amount can be compared across specimen groups | |||
| - Accurate DNA amounts can be measured provided a representative DNA suspension is obtained |