Hi all,
I am currently learning both how to perform relatively simple flow cytometry experiments revolving around the use of propidium iodide (PI) as a DNA marker to look at the cell cycle and also learning/establishing working methods/concentrations etc for cycle synchronisation of my asynchronous cell cultures (U2OS mainly). As I'm fairly new to this whole area, I've hit a few hurdles and would appreciate any advice regarding the below (anything is useful at this point!).
Quick methodology:
a) Harvest cells by trypsinising (~80-90% confluency in flask)
b) Pellet cells
c) Fix in 4% PFA (10min, then pellet and remove)
d) Permeabilise with 0.5% Triton (5min, then pellet and remove)
e) Stain with PI/RNAse and take for analysis
1. I'm not seeing any sort of tell-tale peak suggesting the presence of mitotic cells in my asynchronous cultures, but they definitely grow and double like mad so I know that isn't the case! Every time I harvest/analyse my cells, they always seem to be in G1- and S-phase (see attached picture). The percentages have been manually estimated using FlowJo, and do correct me if I have incorrectly gated/assigned the peaks to the wrong populations. My plot looks like a capital "L" in shape rather than a "hook" shape that I see in pretty much every single other publication which makes me confused... Am I somehow losing my mitotic cells during sample prep/staining? Or is this distribution normal?
2. I've also tried doing both a double-thymidine (2mM) and nocodazole block to synchronise my cells in G1/S and G2/M respectively for future work, though I've had little success... What incubation/blocking times are typically used, as from what I have read 18h seems to be the magic number for the double-thymidine block (with a 9h break in between)? I was wondering if this is just a standard protocol for any cell-line or does this need to be optimised for the specific cell-line in question (given differing cell cycle lengths/drug toxicity etc)?
Apologies for the lengthy post, but I wanted to get all of this down in as much detail as I could. Any and all advice/comments would be greatly appreciated! I want to get this moving asap and move on with applying this onto my other stuff!
Thanks! Jordan
Hey Jordan,
For a very fast-growing cell line it is quite common to have an abundant G1-Phase, if your missing G2/M peak is normal I cannot say. Maybe you can try using standard Nicoletti-Assay protocol. Keep in mind that most of the steps have to be performed on ice since it is a very harsh method for cells.
In addition, try to include your supernatant with dead and detached cells. This gives you information about losing cells into the sub-G1 or >G2/M.
1) Harvest cells (trypsin), pool cells with supernatant, pellet cells at 4°C
2) Wash pellet once with ice-cold PBS
3) Resuspend pellet in 100-250 µL Nicoletti-Buffer (Sodium-Citrate [0,1%], Triton X 100 [0,1%], Propidium Iodide [50 μg/ml], 1x PBS)
4) Incubate for 10 mins at 4°C
5) Measure in FACS
All cell cycle synchronization assays are dependent on the cell line. Therefore, it is very important to know their doubling time. The first block has to be at least 70-80% of doubling time to make sure every cell reaches the blocking point (~16-18 hours). Then you remove thymidine (wash cells thoroughly with PBS) and supply the cells with fresh cell culture media. This recovery ensures that the semi-synchronous cell line re-enters the cell cycle and has to be at least 1,5x S-Phase long and shorter than your doubling time. 9h is a good start. The second block is normally performed 15-25h (1x doubling time).
I would recommend doing a synchronization kinetic once by taking samples every 1-4h and performing the Nicoletti Assay to see which steps of the thymidine or nocodazole block are working and which are not.
I hope it helps!
Best Regards,
Niklas
Dear Jordan!
The analysis using a cytofluorimeter is very approximate and does not provide the information necessary to debug the synchronization technique. Microscopic methods must be used to analyze cells after each treatment. To obtain a synchronous cell population, you need to know not only the doubling time, but also the duration of all phases of the cell cycle of your culture. You can find all the necessary information in my review.
Article Analysis of the Cell Cycle and a Method Employing Synchroniz...
Best wishes,
Rustem Uzbekov
If this is the original data obtained from Flow, I will suggest you to use an extra software for example, ModFit for further analysis, then the G0/G1, S, and G2/M phases will be calculated out by the algorithm in the ModFit software. And the end, you will know whether that one is S phase or G2/M phase or mixed. Thanks
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