Abstract
Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.
Keywords: Flow cytometry, DNA content, Ploidy, Tetraploid neuron, Diploid neuron
Materials and Reagents
Equipment
Software
Procedure
Notes
Acknowledgments
This protocol is adapted from a previous paper by Lopez-Sanchez and Frade (2013). The experimental work was supported by grants from the “Ministerio de Ciencia e Innovación” (BFU2009-07671 and SAF2012-38316) and “Fundación Areces” (CIVP16A1815). Noelia López-Sánchez acknowledges a JAE-Doc contract (JAEDoc026, 2008 call) from the CSIC program “Junta para la Ampliación de Estudios”, co-funded by European Social Fund.
References
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