title: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis.
abstract: Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis. A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.
authors: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation; Sachin.kumar@cchmc.org.
authors: Sachin
authors: S
authors: Kumar
authors: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation; Marie-Dominique.Filippi@cchmc.org.
authors: Marie-Dominique
authors: MD
authors: Filippi
mesh_terms: D002452:Cell Count; D006412:Hematopoietic Stem Cells; D046529:Microscopy, Electron, Transmission; D008867:Microtomy; D013048:Specimen Handling; D013194:Staining and Labeling