Charly, remember, that 95+ % of circulating peripheral blood lymphocytes (in humans or most vertebrates in normal environment, and in SPF or germ-free mice even more) are in resting, i.e., G0 stage. Only very, very few of them, those that were triggered by a cognate antigen within a couple of hours prior to blood taking and not yet exited the bloodstream on their way to whatever lymphatic organ, will have exited the G0 (we show in our papers (Experimental Gerontology 2004) that for human T cells the G0-G1 transition in vitro takes between a day and two (roughly). When you stimulate such restin cells in vitro (with anti-CD3 alone or with anti-CD3+anti-CD28, or with T cell mitogens) they will always respond synchronously for at least first 5-7 division (i.e., up to 5 full days in culture). Thus it isnot necessary to stop them in tracks with hydroxyurea, as they do start from a stopped stage. Not so if you isolate the lymphocytes from spleen or lymph nodes of a mouse (or - say - from human nodes or tonsils); there you will find antigen responding cells in many different staghes of cell cycle, so you must use hydroxyurea or alike to synchronize them.

Dear Charly, T lymphocytes can be easily synchronised by incubation in the medium containing 2 mM hydroxyurea. This reagent induces G1/S block. The cells will be synchronised at this stage and after washing cell cycle will continue.

Thank you very much! I will give it a go.

make sure you do that when most of the T cells are in cycle.

Charly, remember, that 95+ % of circulating peripheral blood lymphocytes (in humans or most vertebrates in normal environment, and in SPF or germ-free mice even more) are in resting, i.e., G0 stage. Only very, very few of them, those that were triggered by a cognate antigen within a couple of hours prior to blood taking and not yet exited the bloodstream on their way to whatever lymphatic organ, will have exited the G0 (we show in our papers (Experimental Gerontology 2004) that for human T cells the G0-G1 transition in vitro takes between a day and two (roughly). When you stimulate such restin cells in vitro (with anti-CD3 alone or with anti-CD3+anti-CD28, or with T cell mitogens) they will always respond synchronously for at least first 5-7 division (i.e., up to 5 full days in culture). Thus it isnot necessary to stop them in tracks with hydroxyurea, as they do start from a stopped stage. Not so if you isolate the lymphocytes from spleen or lymph nodes of a mouse (or - say - from human nodes or tonsils); there you will find antigen responding cells in many different staghes of cell cycle, so you must use hydroxyurea or alike to synchronize them.

That is certainly a very interesting point to remember. These will be splenocytes so I should probably synchronise them, but we are also going to be doing some work with human PBMCs so I will definitely bear that in mind!

I do not agree with Jacek. I think, you will need to synchronise them anyway. Evidently, activated T lymphocytes (even activated from resting stage) can not be recognized as synchronised cells due to differences in durations of G0/G1 transition as well as cell cycle for individual cells.

Dmitry, it does not matter how long the resting T is in G0; it is stopped, so when stimulated all responding T's will start at the same time. As we show, duration of G0/G1 transition depends on which population or subpopulation you study (e.g., CD4+CD28- take longer to start their first G1 than CD4+CD28+ from the same individual, and cells of older individuals take longer to start than those from younger ones. Variation of G0/G1 transition in the circulating PBL of an individula is negligible. Hydroxyurea would remove the problem of G0/G1 duration differences only if you would take the cells and stimulate them first, separate the responding ones and then expose to HU (to have all available dividing cells stopped at G1/S initially). Still, HU treatment would not help for variation in cell cycle duration (after a single cycle the initially synchronized cells differing in the individual; cell cycle length would get out of synch already after a single division). So, for practical use, the effect of HU synchronization would not surpass using the resting peripheral blood lymphocytes starting from G0 (which, additionally, eliminates a potential interference of HU in the experimental setup).

Dear Jacek! Please, be realistic. Blasts in the stimulated PBL culture are different in sizes, to see metaphases you will need to use colchicine,..etc. They are NOT synchronised!

Dear Dmitry, please don't shout. True, blasts are of different sizes (as can be nicely seen in a FSC/SSC FACS plot) but, as the same technique tells you, a blast does not have to be the largest cell around to enter mitosis; in other words, different sizes of blasts do not indicate they are in grossly different stages of division cycle. Actually, we do see practically the same size variability in the anti-CD3 stimulated, HU-stopped blasts . You use colchicine not to see metaphases, but to see many of them at the same time (as the average mitotic evolution of a lymphocyte is about one hour or less, it is true that at any given time without colchicine or alike you would only see just a couple percent metaphasic cells; this would rise a couple of times if you would count all cells in different stages of mitosis). Initial synchronization of human peripheral blood lympjhocytes when stimulated in vitro with PHA was very recently stressed by Susan H Ford (Mutagenicity 2013 1-8, doi:10.1093/mutage/get004).

Thank you, Jacek! Very useful reading. Good luck in your studies!

And same to you, Dmitry!

The best thing to do is to put the cells on a centrifuge with an elutriation rotor, they come out enriched on the different phases of the cell cycle. You need somebody who knows how to work it but it will be the best and you can get a large number of cells that can undergo at least one cycle synchronized.

The second best thing would be to make a short pulse with BrdU (about 30 min). They will be not synchronized, but the only cells that will be labeled are the ones that were in S during the labeling time, and these you can follow by FACS for at least two cycles. Usually keep synchronized for at least one cycle.

The third thing I would do is to transfect these cells with pFUCCI-S/G2/M Green and pFucci-G1 Orange, so hat you can see the cells in green during S/G2/M and in Orange when they are in G1.

What I would not do, if you want to be serious about your results is to use drugs.

You could also label the cells with Hoescht 33342 and analyze them depending on the phase of the cell cycle they are, but the three above, and in such order are my prefered ones.

Best

Jose A.

Jose A, good comments. Yet elutriation is working nicely only if you have plenty of unsynchronized cycling cells (say Jurkat T cell leukemia line, or primarylymphocytes kept long( many PDs) in culture). I like your idea of marking specific stage of the cell cycle and following marked cells only :-) yet for this purpose I would not use anything binding DNA, if I want tu study the cells' functions. Hoechst dyes nicely stain DNA allowing for pretty visualization of stages of the mitotic cycle, yet treated cells cannot be used for functional studies.

we have used them for the analysis of IL-2 deprivation induced apoptosis, demonstrating that the presence of some chemokines abrogate the apoptosis. These results were latter on verified by limiting dilution analyses...... It worked well for us. We have done similar things with primary cells.....

Usually the number of cells for elutriations is not a problem, even with human PBMCs, you can get 10^9 from a buffy coat, with that amount you can do 10 elutriations.... the tratments come after....

Hi Charly,

Please find attached a protocol including three different synchronization methods. All three are used to synchronize in differnt phases of the cell cycle.

Usually at least one should also work for your cells!

Thanks very much all for your help!

Hi Charly,

I have had worked with HT-29 cells, a human adenocarcinoma colon cell line. We don't arrest of the cell cycle, but we use colchicine to produce G2/M cycle arrest as control and to considerate where the different phases are. In addition, some groups in my building do the synchronization of the cultures (other cell lines) adding only free fetal medium for several hours ( I don´t remember very well, 2h?), and then they wash with PBS and add fresh/completed medium and the drug to study its effect.

Other group works with hamters cells and do the syncronisation with colchicine (0.1 or 0.2 microg per mL for 2 or 4 h) and then carry on the protocol: add the drug...

I hope to help you

Best regards !!!

Below article may useful in the troubleshoot.

http://www-personal.umich.edu/~cooper/Cell.pdf

Just use a starving medium for a few hours (12). We have tryied with 1% of serum (heat inactivated FBS) just with glutamine 10% (RPMI).