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Background

Proliferation & Differentiation

HemoGenix® distinguishes between cell proliferation and differentiation assay.

 

  • 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 by a drug or other agent or perturbation, 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 (e.g. a clonal methylcellulose assay) assumes proliferation, but does not measure proliferation.
  • 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

HemoLIGHT™-96

MSCLIGHT™-96

ImmunoLIGHT™-96

STEMLight™-96

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

HemoFLUOR™-96

MSCFluor™-96

ImmunoFLUOR™-96

STEMFluor™-96

Bioluminescence ATP as a substrate for a luciferin/luciferase reaction to produce bioluminescence in the form of light detected by a plate luminometer

CAMEO™-96

HALO®-96

ImmunoGlo™-96

MSCGlo™-96

STEMGlo™-96

 

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

ColonyGro™

CAMEO™-4

CAMEO™-96

STEMClone™