Methods for age determination and sorting of cells according to their age

Note

• Technical description
• precision,
• limitations,
• results of experiments done with these techniques.
• Comprehensive bibliography

Yeast s. cerevisiae

• replicative life span:

first done by mortimer (1959): micromanipulation: throwing away the daugther cell

• Separation of Old and younger cells:

1/ centrifugation: egilmez,1989 young buds and cells are supernatant., synchro, then centri...etc (egilmez et al, 1989, 1990) identification of genes which expression level change with age.

2/ biotin labelling of initial mothers: h Biotin covalently attach to primary amines like the α-amino of Lysine.

Biotinylated surface proteins are retained in mothers but are not found in their daughters,because the daughters’ cell walls are newly synthesized.then fluorescent Avidin is added and cells sorted by facs. For large scale, magnetic beads assisted purification. Smeal et al, 1996 quite efficient toxic. The authors find a loss in the silencing by SIR proteins in old cells.

3/chen and contreras used microbeads (50 nm diameter). Because of their small size, microbeads did not affect cell physiology and therefore bead detachment was not necessary. Then WGA to stain chitin, and FACS. Not so precise however.

3/ Then they all count bud scars to determine the average age of the isolated population. Not really sharp isolation, event with double purification.

Old phenotype: Bigger, scars with chitin, symetric cell division, cell cycle slows.

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Mammalian cells

Primary cells, non immortal.mostly human fibroblasts. Hayflick limit (hayflick, 1961). in vitro, senescence due to a telomeres shortening.

Note the technical limitation allows only to separate youngvs senescent cells. No intermediates, no clear dimension of which gradual processes can happen..

• Methods

Actually, at a given time, a population of Human fibroblasts is highly heterogenous; It's a mix of senescent and non senescent cells, and the number of senescent cells increase with population doubling. Not all cells enter senescence at the same time. So how do people manage to obtain "pure populations" of senescent cells?

Senescent phenotype:: to be expanded and detailled with references.

cell and nuclear sizer (big),shape, production of ROS, Beta galactosidase activity, age pigment accumulation (lipofuscin), gene expression pattern, mitochondrial function.

Methods for Cell Sorting of Young and Senescent Cells: João F. Passos and Thomas von Zglinicki (from Methods in Molecular Biology: Biological Aging: Methods and Protocols Edited by: T. O. Tollefsbol © Humana Press Inc., Totowa, NJ)

Fixed cells

staining and FACS for different markers:

• proteins upregulated in senescent cells: p16, p21, γ-H2AX.
• Failure to replicate: brdu incorporation, negative selection.
• staining for SA-β-galactosidase active at pH6 only in senescent cells (Dimri et al, 1995)

live cells

It's better if it's alive!

• Size+ autofluorescence

Cells get bigger and lipofuscin, the autofluorescent pigment accumulate with age.

• Reactive Oxygen Species levels

DHR 123 fluorescent sensor of ROS (link)

• Mitochondrial membrane potential

JC-1 probe sensitive to MMP (link

• Genetically encoded fluorescent reporters

GFP under the control of a p21 promoter: Cheng et al PMID 16779499

Organisms

• c.elegans
• drosophila/mice: detecting somatic using lacZ Garcia et al., 2007

the authors insert copies of the lacZ gene in the germline of fly or mice. Then when the animal age they collect the tissue they're interested in, purify DNA, excise the plamids containing lacZ and transformed into a special e.coli strain allowing selection for lacZ mutants. Then they amplify colonies and characterize the type of mutations that occured.