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Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

Kabani S, Waterfall M, Matthews KR.

Mol Biochem Parasitol. 2010 Jan;169(1):59-62. doi: 10.1016/j.molbiopara.2009.08.008.

Sarah and Martin did an excellent job of optimizing sorting for bloodstream form T.brucei.

Hi Catarina,

As far as I'm aware, propidium iodide only enters live BSF cells and binds to DNA very slowly - In the past I've used it in an assay to determine when cells actually die (see attached paper). What might help is if you use a stain like DAPI, which is rapidly accumulated by live BSF cells. However, both of these compounds will ultimately be lethal to the cells as they bind DNA so maybe another cellular stain entirely would be better, or perhaps a GFP-fusion protein under the control of a life-cycle stage specific regulator if you're trying to sort BSF from differentiated PCF/Stumpies?

Check out:

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

Kabani S, Waterfall M, Matthews KR.

Mol Biochem Parasitol. 2010 Jan;169(1):59-62. doi: 10.1016/j.molbiopara.2009.08.008.

Sarah and Martin did an excellent job of optimizing sorting for bloodstream form T.brucei.

Hi both,

Thank you for the prompt replies!

I'm fixing them anyway, I want to sort them and then extract gDNA. I'll take a look on both then! I'm sure I've checked Kabani et al 2010 before, and I'll take a look again, and if it still does not work with PI, I'll try and see if I can get similar results using the Vybrant DyeCycle Violet. Thanks again! Best wishes!

I am not sure why you would need to sort procyclics from bloodstream forms. And when the need occurs it may be much easier to used culture conditions that are selective. Long/slender BSF do not survive standard procyclic growth conditions, so you automatically will have a procyclic-only culture.

As for the propidium iodide staining, propidium does not appreciably enter live trypanosomes, the parasite does not have a transporter for this molecule and it does not diffuse across membreanes. Thus, it is actually used as death marker for trypanosomes: only those trypanosomes with a breached plasma membrane will be stained with propidium iodide. This sorting PI-stained trypanosomes is selecting for dead parasites - or more accurately, those with a permeable plasma membrane. I attach some papers where PI staining is used as cell death indicator and/or in FACS to assess membrane permeability:

- Gould MK, Vu XL, Seebeck T, and De Koning HP (2008) Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay. Anal Biochem 382:87-93

- Ibrahim HM, Al-Salabi MI, El Sabbagh N, Quashie NB, Alkhaldi AA, Escale R, Smith TK, Vial HJ, and De Koning HP (2011) Symmetrical choline-derived dications display strong anti-kinetoplastid activity. J Antimicrob Chemother 66:111-125

This is the other paper I mention in my first comment.

Hi Catarina,

When I've done FACS analysis on BSF T. brucei the following has worked for me...

1. Resuspend 10^7 cells in 300 ul cold PBS and add 700 ul ice cold methanol; ice 10', then 4C overnight

2. Harvest cells 200g (~1500 rpm in a standard microfuge) for 10' at 4C

3. Resuspend in 1 ml PBS; transfer 10^6 cells to PBS in a final volume of 1 ml

4. Add 10 ug/ml RNAseA, then 10 ug/ml PI; place at 37C for 45' (in the dark)

5. Perform FACS analysis within 2 hours

I've found that leaving the cells in methanol for longer than 24 hours or delaying the FACS analysis leads to sub-standard results. Following the above protocol gives plenty of cells for standard analysis, though I've not then gone on to do sorting. Using this protocol, the cells may not be robust enough to maintain their integrity during the sorting process, so you may need to investigate alternative fixing protocols (PFA?). Hope this helps, good luck with solving the problem!

Hi Catarina,

When you mentioned PI I assumed you were analysing fixed cells. Is this not the case? If not, then Pegine's suggestion is definitely worth following up.

Hi all,

Sorry Harry if I did not made myself clear at all, I've sorted PCFs successfully and now I'm trying to sort BSFs, I'm not trying to get BSFs from PCFs at all. I should have explained better, I'm sorry.

In both cases I'm interested in sorting fixed cells, in this case in 70% methanol, and for procyclics this has worked very well with PI. My problem with BSFs is that by applying the same protocol I end up losing a huge amount of cells (e.g. starting with the same number of cells, I was getting a rate of 4.000 evt/sec with the PCF and got 40 evt/sec with the BSFs), which makes sorting not feasible. I'll take a look at the papers you've suggested.

Hi Sam, I've used pretty much the protocol you've mentioned, with the exception that we always use PBS with 5mM EDTA to avoid clumping. This has worked very well with procyclics, but as I mentioned, is not being good at all for BSFs. Nevertheless, I've left them longer in methanol, so I wonder whether that might be one of the factors. What I usually do is to collect the cells, fix them and leave them at least overnight in methanol at 4C, then for up to a couple of weeks, whenever I need them for the sortings, I perform the staining protocol as you said. I think I'll start troubleshooting by leaving them in methanol for different periods of time and check if I'm loosing cells with the longer presence in methanol.

Thank you all for your suggestions, I'll take a careful look at the papers and see if I can get the BSF sorting rate high enough to make it feasible!

Best wishes,

Cat

The adult form of thripanosoma is 25-30 micron,are you sure you do not plug the tube sorter?

Hi Anna,

I don't think so, I can see events passing through and I don't see any clumping. The FACSAria technician is always present and she checked up the machine during and after and she has not mentioned anything suggesting that the tube was blocked. We're always very careful about that. Nevertheless, I'll extra attention this time and check with her whether any plugging is happening.

Hi Catarina,

If you are sorting living cells, PI is not good for DNA staining. Otherwise, the fluorescence detector may be not sensitive enough to detect the PI stained nucleus. You may try increase the gain of the amplifier of the FL2 channel (BD incorp.) to enhance the detector sensitivity, or try incubate cells with PI and RNase for longer.

Good luck!

Hi Shanhui,

Thanks for your reply.

I'm using fixed cells, and the protocol I've used (pretty much the one Sam Alsford just mentioned in one of the replies above) works perfectly for procyclic cells, the insect stage of the parasite. My problem is that when applying it to blood stream form cells of T. brucei, I lose most of my cells, but the ones that pass through the sorting machine have a good staining. So I don't think the problem is PI not staining, but it might be something to do with the differences existent between the two developmental stages of the parasite that might render the blood stream form cells more susceptible to the protocol than the procyclic ones.

I was just wondering if other people were having the same problem, and I'm sorry I did not made myself clear that the cells were fixed.

Thanks again, best whishes!

Hi Catarina,

We have done this technique, with BSF, quite routinely here, most recently I think Abdulsalam Alkhaldi in my group, but he has now left. Below I paste the protocol from his thesis for your attention:

2.7 Cell cycle using flow cytometry

Flow cytometry was used as described by (Hammarton et al., 2003) to study the effects of test compounds on DNA content in Trypanosoma brucei brucei. The cell density was adjusted to 1×106 cells/ml with and without test compounds for the duration of the experiment. 1 ml of sample was transferred at each time point into microfuge tubes and centrifuged at 2500 rpm for 10 min at 4°C, then resuspended and fixed in 1 ml of 70% methanol and 30% PBS and left at 4 °C overnight. After storage, the samples were washed twice with 1 ml of PBS and subsequently resuspended in 1 ml PBS containing propidium iodide and RNase A (both at 10 µg/ml), and incubated at 37 ºC for 45 minutes while protected from light. Samples were analysed by Becton Dickinson FACSCalibur using the FL2-Area detector and CellQuest software. ModFit LT software was used to quantify the flow cytometry results.