Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells

Colony formation assays measure cells’ proliferative capacity to grow into colonies.

First, seed bone marrow-derived cell suspension comprising erythrocytes and mononuclear cells, including mesenchymal stem cells or MSCs, in media on a culture plate and incubate. MSCs, adhere to the plate bottom while erythrocytes and mononuclear cells remain in suspension and are discarded.

Add fresh media containing growth factors to support MSC survival. Depending on their individual proliferative potential, MSCs replicate to form colonies.

Next, treat the colonies with methanol. Methanol permeabilizes the cells, displaces water from the intracellular environment, and denatures proteins, preserving cellular architecture.

Incubate the fixed colonies with Giemsa staining solution - a mixture of oxidation products of methylene blue, primarily azure B, a basic dye, and eosin Y, an acidic dye. Upon diffusion into the cells, positively-charged azure B dimers bind via intercalation to negatively-charged DNA in the nucleus, imparting a blue stain.

Negatively-charged eosin Y monomers then interact with un-neutralized positive charges on bound azure B dimers, forming an ‘eosin-Y-azure-B complex’, changing the coloration of blue-stained DNA to purple.

In the ribosome-rich cytoplasm, azure B binds to negatively-charged RNA, but reduced penetration of eosin Y into highly-dense ribosomes, prevents eosin-Y-azure-B complex formation, staining RNA blue.

Now, wash the plate with buffer to remove excess stain and air-dry. Count the number of colonies, which appear as clusters of cells with purple nuclei and blue cytoplasm.