Background
Proliferation & Differentiation
All cell-based assays will require the detection of either proliferation or differentiation or a specific aspect of the proliferation or differentiation process. The interpretation and conclusions obtained from the results will depend upon using the correct assay for the goal of the experiment or study. It is therefore important to understand proliferation and differentiation, their relationship and the characteristics of proliferation and differentiation assays.
What is Proliferation and What is Differentiation?
Proliferation may be defined as the expansion of cells by the continuous division of single cells
into two daughter cells.
Differentiation is the process whereby an undifferentiated cell, e.g. a stem cell, acquires the
features of a specialized cell.
The Relationship Between Proliferation and Differentiation
- Proliferation occurs prior to differentiation.
- Without proliferation, differentiation would not occur.
- Differentiation is a default program requiring prior proliferation. But the corollary is not true.
- If proliferation is affected, then differentiation will also be affected, but not visa versa.
Characteristics of Proliferation and Differentiation Assays
- Although the processes of proliferation and differentiation overlap, from an assay viewpoint, they are two separate processes.
- A proliferation assay cannot measure differentiation and a differentiation assay cannot measure proliferation, because proliferation and differentiation require different readouts.
- Although cells must undergo proliferation to differentiate, an assay that detects a differentiation process assumes proliferation, but does not measure proliferation.
- In other words, it is NOT possible to measure proliferation and differentiation in the same assay using the same readout.
- In general, proliferation assays are quantitative since they are usually dependent upon the measurement of a biochemical reaction or interaction. They can therefore be easily calibrated, standardized and validated.
- In general, differentiation assays are subjective or semi-quantitative at best and are therefore difficult, if not impossible, to calibrate, standardize and validate.
Proliferation Assay Readouts
With the exception of basic cell counting, all proliferation assays also detect cell viability, cell functionality and cell number. There are essentially 5 types of proliferation assays:
| Assay Type | Detection Method | HemoGenix® Assay Examples |
|---|---|---|
| Cell counting | Manual or electronic | |
| Radioactive labeling | Autoradiography, scintillation counting | Not available |
| Colorimetric/Absorbance | MTS Tetrazolium reduction to soluble formazan and measurement at 490nm in an absorbance plate reader | |
| Fluorescence | Cleavage of GF-AFC substrate by living cell protease activity to produce fluorescence excited at 380-400nm and emission at 505nm in a fluorescence plate reader | |
| Bioluminescence | ATP as a substrate for a luciferin/luciferase reaction to produce bioluminescence in the form of light detected by a plate luminometer |
Differentiation Assay Readouts
| Assay Type | Detection Method | HemoGenix® Assay Examples |
|---|---|---|
| Molecular Expression | PCR | Can be multiplexed with any HemoGenix® assay platform |
| Intracellular expression | Image analysis, ELISA, flow cytometry | Can be multiplexed with any HemoGenix® assay platform |
| Extracellular membrane expression | Image analysis, ELISA, flow cytometry | Can be multiplexed with any HemoGenix® assay platform |
| Colony-forming assays | Manual, image analysis |
Choosing the correct assay for the study goals can reduce the risk of falsely interpreting results