These guides are written primarily for HeLa cells, but it should be possible to extend these to other cell lines with a bit of optimisation.
1) Make sure your cells are happy !
2) Aim for around 75-85% confluence at the time of release.
3) Try and keep everything close to 37ºC, including media, washing media, PBS, TC hood, centrifuges and avoid having the cells out of the incubator for long periods… i.e. work quickly and be organised before you start.
4) Avoid excessive amounts of media on the plate. Cells need to condition the media and the more volume the longer it will take. For a 10cm plate 5-7ml is ideal depending on the timing. Thus short releases (less than 12 hours) use 5ml, longer use 6-7ml.
5) Always try and make sure your cells are very well spread out on the plate. Clumps and areas of very high or low density will reduce your synchrony.
Here you will block cells in late G1 early S phase usually for around 18-28 hours. This time is roughly equivalent to how long the doubling time is for the cell line. For HeLa’s 20-24 hours is normally used. Cells will roughly take around 6 hours to complete S phase, 1-2 hours for G2, and around 1 hour for mitosis. Thus roughly:
S phase = 2-6 hours after release.
G2 = 7-8 hours
Mitosis = 9-10 hours
G1= 11+ hours
1) Seed up asynchronously growing cells on your desired plate. Your seeding will depend on when you plan to block the cells. You can block at the time of seeding, once they have resettled or the next day. Generally you will want about 50-60% density.
2.5mM (Update we have dropped to using 1mM, best to do a check with your batch of HeLa’s to determine the optimal dose) Thymidine, which depletes the cells of deoxycytidine triphosphate. NB: Thymidine is not very soluble in water so make it up to a stock [100mM] in PBS, and make sure you use PBS not water will need to add 25µl/ml of media.
3) Wait 20-24h, to arrest the majority of cells in G1/S. NB: you will always still have the odd few cells in mitosis… but thats ok.
4) Wash the cells 3x with pre-warmed PBS or Media. I have found that media sometimes gives slightly better results, although this is the more expensive option.
5) Add back fresh media, with 25µM 2′-Deoxycytidine (Santa Cruz #sc-231247), to replenish the depleted pools, and promote timely entry into S phase. NB some protocols suggest adding 25µM of Thymidine as well, this may help improve the releases slightly.
1) After the first release (step 4 above), wait 8-10 hours and then block cells again with 2.5mM Thymidine for an addition 16 hours.
2) Release as before (step 5 above).
NB: a double block is preferred when you require a very tight synchrony and time points from each different cell cycle phase for biochemistry. However, it does add a extra layer of complexity and if not done perfectly can result in a worse synchrony than a single block.
Similar to thymidine although you use 2mM to block the cells. Also this can only be done as a single block, and no 2′-Deoxycytidine is needed in the release media.
The downside to the method is that some cell lines (e.g. U2OS) will start to over-duplicate their centrosomes if left in Hydroxyurea for too long.
Late G2 Block
This method is excellent for enriching cells in mitosis for either IF or movies.
RO-3306 (Cdk1 inhibitor)
Although a single block can work, best results are achieved by first blocking cells in G1/S with Thymidine or HU.
1) Release cells from G1/S as per instructions above.
2) Add 10µM of RO3306 to cells, ideally 4-6 hours post release to allow cells to get thru S phase.
3) Wait until the majority of cells have reached late G2. This is usually around 12h post release from G1/S.
4) Wash out the drug very well, with at least 3x washes with media or PBS.
5) Add back fresh media. Cells should begin entering Prophase within about 15-30 min of release. Metaphase peaks around 30-60 min and most cells should have completed anaphase/telophase by 90-120 min.
Here is a movie showing a best case synchrony, where around 90% of cells undergo mitosis. Typically, you should expect to see 50-60%.
This method is excellent for doing biochemistry on mitosis as it allows for highly enriched samples with a tight synchrony. It can also be used for movies if great care is take during the washing stage not to wash the cells off the plate. It is not suitable for IF.
Like RO3306, you can use just a single block with Nocodazole for 20-24 hours, but this will result in an increase in the level of death and better results are achieved by doing a pre-sync with thymidine or another G1/S blocker. NB: cells with a functional Chfr/Antephase checkpoint will delay for significant time during late G2 early prophase. HeLa cells do not have a Antephase checkpoint, but you may still notice a 1-2 hour delay in mitotic entry in response to Nocodazole.
1) After release from G1/S add Nocodazole at the desired concentration (25-3000ng/ml) ideally 4-6 hours after release, although you can add it straight away if you’re lazy. If you plan on releasing cells from the Nocodazole arrest then use 25-50ng/ml. If you are only interested in blocking cells use 100ng/ml. If you want to completely depolymerise microtubules then use 1-3µg/ml.
2) Wait 12-14 hours after release, this should be sufficient for the majority of cells (80-90%) to block in Pro-Metaphase (P-M). You can easily see this by comparing the number of rounded up cells (P-M), to flat attached cells, likely G2.
3) For Biochemistry, recover media and floating cells from plate and put into a falcon tube. Bang the plate several times to help detach mitotic cells. Add a small amount of fresh (warm) media to recover detached cells. Repeat this once more. Check plate under a microscope that you still have interphase cells attached, and have recovered the majority of P-M cells.
4) You now have a nice highly enriched sample of pro-metaphase cells.
5) If you want to release, then gently spin cells down for 2-3 minutes at low speed (usually 1000rpm is sufficient). Remove media, gently resuspend pelleted cells with excess of fresh media (without any Noc in it). Repeat 1-2 times. NB: cells are very fragile at this stage, excessive washing can damage them leading to a poor release. Thus its a balancing act between washing away enough Noc to allow cells to recommence Mitosis. Generally cells will start arresting at doses of around 5ng/ml of Noc.
6) For movies, do not shake off the cells. Be very very very gentle with the plate, remove the media, and slowly add back fresh media. Repeat 2-3 times. Then immediately start filming your cells.
Grow cells in DMEM (Sigma #D9443) without L-Arg, L-Leu, L-Lys !
This DMEM is low glucose 1000mg/L so you will need to add and extra 3000mg/L to make it unto 4000mg/L in total. Thus 15ml of the 10% Glucose solution is needed for 500ml of DMEM.
Need to add 50ml of Mass Spec Grade Serum for final [conc.] of 10
1X Normal [Conc.] for DMEM media
L-Arg = 398 µM
L-Leu = 802 µM
L-Lys = 798 µM
From testing HeLa cells grow well and have very high incorporation rate at 1/4 to 1/8 of these levels. To play it safe we have been using 1/4. Thus:
L-Arg = 100µM (Light and Heavy)
L-Leu = 200µM (Only Light needed)
L-Lys = 200µM (Light and Heavy)
Grow cells for at least 5 doublings to ensure complete labelling. As a rough guide, if you Start on Monday, you will be ready by the following monday !