I have read papers that describe methods for long-term primary neuron culture but I have not seen convincing western blots showing consistent changes in any protein expression with the progress of aging.
I know that aging is a complex process, but I would like to somehow test if my long-term cultures are actually going through an aging-like process.
There are several classic senesence markers that appear to increase in many cell types. I'd recommend going with the classics p16INK4 (CDKN2A) and p21CIP1 (CDKN1A). You could also try CD44. All three are significantly increased in long term cultures of primary human corneal endothelial cells, which just so happen to originate from the neural crest. If you culture just about any primary cell long enough, it will eventually enter replicative senescence (i.e., it will show signs of aging). Another feature is cell morphology, senescent (aged) cells have a tendency to increase in size and flatten out, quite significantly. You may also consider assaying for GLB1 activity (increased activity in many sensescent cell types).
Hi Jiyeon，
Suppose many markers can be found if searching proteins levels change with aging, I do suggest one crucial signaling-mTOR pathway. The mTORC1 activity is downregulated along with cortical neurons maturation an aging. The phosphorylated S6 level was validated to decreased with aging, you may try it. Western blot and immune staining both work. Good luck.
Hi Jiyeon,
Long term culture could contribute to aging process of neurons. The senescent cells are one of hallmark of aging and a key process of aging in cell culture and tissue. The identification of senescent cells is based on an increase level of lysosomal β-galactosidase activity. β-galactosidase activity is detected by Senescence-associated β-galactosidase staining (Senescence assay). The senescence-associated secretory phenotype (SASP) factors are a biomarker of aging cells including ROS, NO, IL1, IL7, IL8, IL13, IL15, TNF, and PGE2 etc. which contribute to aging. Senescent cells slow proliferation and secrete pro-inflammatory cytokines, chemokines, and extracellular matrix proteases, which, together, form the SASP. So, you can define these factors as biomarker of aging. Hope this help
Best,
Ruchee
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