I treat quiescent murine B cells with some stimulants to make them divide. They enter into the cell cycle and they divide for about 10 days and then stop dividing. At any point, if the stimulants are withdrawn, the cells die (likely by apoptosis) due to lack of survival signals. Whereas if the cells are maintained with stimulants, they are alive for longer. Since these are the very same stimulants I use for making them get out of quiescence, I do not know the state of the cells because they are neither dividing not dying after about 10 days.
How will I confirm if they are back to quiescence again? Or they are terminally differentiated? Or they are (replicative) senescent?
If I am not wrong, the terminally differentiated state is plasma cell (there could be others) and they have some distinct morphology like more cytoplasm and more elongated. So maybe you check them under microscope). You can also assay for secreted antibody using Fc ELISA? Plasma cell produce a lot of Ab. B cells maybe some but a lot less.
If senescent then cell might produce some Senescent Bete galactosidase so you might stain for them! But I think it should not be senescent (unless your cell line have many passages). Most primary cells only become senescent after many decades!
Hi Abel,
I am dealing with a murine system. The treatment I provide actually inhibits terminal differentiation to plasma cells. I shall eventually check markers and secreted antibodies though. But talking about the state of cells, I am confused, because, if the cells were quiescent, they should be responding to stimuli and stay alive in the absence of stimuli. In my case, they die without the signal and don't proliferate in the presence of the signal. Since they might have crossed 10-12 divisions by 10 days, I was thinking they could have undergone replicative senescence (Hayflick's limit). How likely do you think is this possibility? And may I know the reason/logic behind primary cells becoming senescent only after decades?
Thanks!
Sorry I was thinking you were working with human cells.
Since human HSCs dont go senescent after the person is old that means the B cells also cannot be senescent unless the HSCs themselves are aged.im aware in humans during differentiation B cells undergo telomere lengthening. Might be why memory cells are not senescent after having undergone clonal expansion.
Weng, N., Granger, L., & Hodes, R. J. (1997). Telomere lengthening and telomerase activation during human B cell differentiation. Proceedings of the National Academy of Sciences of the United States of America, 94(20), 10827–10832.
I think in humans, B cells die when not given survival signals, whether or not theyre senescent or quiescent.Depends on what kind of survival signals you give.
With mice I dont know how long the telomeres last or if the same phenonemon occurs.Sorry Im not a mouse guy!
I forgot to mention the organism in the original question, sorry about that. I'll think along the lines you have mentioned for the human counterparts. Thanks.
Hi Prasad,
First look at morphology of nucleus in your cells and do H2AX staining to see whether you have activation of DDR in your cells and do immunoblot with that lysates.
You can use some markers to differentiate a cell from apoptosis
1. stain with H2AX and try to look for SAHF formation in your cells.
2. Do beta gal assay.
3. Look at the expressions of nulcear lamins, this is the only marker available to differentiate a cell from quiescence and senescence.Nulcear lamins doesnt decline in quiescence cells.
i hope this information would be useful for you to see whether the cell is quiescent or senescent.
Hi Hari,
Thanks for the suggestions. Unfortunately, since my cells are in a suspension, I cannot observe extensions like in adherent cells. And since they are lymphocytes, their nucleus to cytoplasmic ratio is already large. I will try to work the assays out though.
Hi Prasad I am working on senescence and I can help you if you need some information regarding senescence
That's great. I will try to perform some of the experiments and get back.
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