The isotropic fractionator
Herculano-Houzel & Lent, J Neurosci (2005)
This is a novel, non-stereological method for counting numbers of cells (neuronal or non-neuronal) in any dissectable brain structure, or in the brain as a whole, once the tissue of interest is dissolved by mechanical dissociation in detergent and transformed into an isotropic suspension of known volume that contains all the cell nuclei present in the original tissue. The method is based on the assumption that every cell in the brain has one and only one nucleus: as long as this is true, counting nuclei is equivalent to counting cells.
No extensive training is required to use the isotropic fractionator: basic microscopy, immunocytochemistry and cell counting skills suffice, and the necessary equipment amounts to an upright fluorescence microscope, a glass tissue grinder and a hemocytometer, other than antibodies that recognize nuclear antigens.
Counting from the moment when the tissue is well-fixed and ready to be processed, total numbers of neurons and non-neuronal cells in the tissue can be determined in less than 24 hours. It takes 20 min to homogenize up to 1 g of tissue, and, for the trained eye, another 30 min to estimate total number of cells in the tissue under the microscope. Once a sample of the suspension is stained with anti-NeuN antibody, in another 30 minutes the fraction of nuclei that are neuronal is determined under the microscope, and the non-neuronal fraction is derived by subtraction.
Counting numbers of cells in four samples of the suspension yields average estimates that have a CV typically below 0.15, and often below 0.10. CVs above that indicate that the suspension is not isotropic and must be further mixed by agitation before recounting.
All that is required, besides a fluorescence microscope, a hemocytometer and a glass tissue grinder, are the adequate antibodies and DAPI for visualisation of all cell nuclei.