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Cyanobacteria and microalgae

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OLIVIASP

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Posts: 6

Joined: Fri Jun 17, 2016 9:04 am

Post Fri Jul 12, 2019 11:35 am

Cyanobacteria and microalgae

Dear all,

Does anyone work with cyanobacteria and microalgae in Cytof? We think that we won´t have problems with microalgae altough we know they are quite small (not viewing individuals events) but cyanobacteria apart from their shape (different compare a human cell and a bite narrow) they are big and we think the instrument can´t ionize them in a correct way. Regarding to the Iridium, cyanobacteria are procariote and we want to know if the protocol to label the nucleus is the same as a human cell.

Thank you in advance.
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jul 12, 2019 2:00 pm

Re: Cyanobacteria and microalgae

Hi Olivia,

I'm not aware of anyone having looked at algae before. However, there are at least 3 papers for CyTOF on bacteria (E.coli).

In the paper I published, we had problems getting the Ir stain to work well....just not much signal. Whether this was a perm issue or the fact that the E.coli genome is 1000x smaller than human and therefore has a lot less DNA, we never firmly concluded. Instead, we developed a membrane stain that worked well. For your algae, keep genome size in mind, but otherwise you won't know til you try....

What is the size range of your targets (largest and smallest)? There is an upper limit to how big something can be and still be completely ionized. For solid inorganic nanoparticles, it's in the 100nm range, but I don't know of anyone has formally tested it in terms of cells (bags of water).....most of the time we're focused on clogging of the instrument with huge cells (eg adipocytes) rather than trying to determine whether they're fully ionized.


Mike
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OLIVIASP

Participant

Posts: 6

Joined: Fri Jun 17, 2016 9:04 am

Post Sun Jul 14, 2019 8:56 am

Re: Cyanobacteria and microalgae

Hello Mike,

The research wants to work with cyanobacterias (0.8 microns x 30-40 microns) they are prokariote. Microalgae are eukariotyc with a size 2x2 microns, very small. For the procariota we will try the same in the paper... a membrane stain could be a great idea.

We normally run human cells in our Cytof, we know about the problems with clogs and I am sure it will be a nightmare with cyanobacterias. Also we must run a quick check first to control for environmental contaminants (i.e, mercury, iodine).

Thank you Mike,

Olivia.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jul 15, 2019 2:51 pm

Re: Cyanobacteria and microalgae

Hi Olivia,

1. The 30-40 micron length might be challenging. As a reminder, the internal diameter of the nebulizer is on the order of 100 micron. At the very least, dilute your sample so it's less likely to get cells stuck together.
2. The membrane stain worked well for us. Here's our paper on it: https://doi.org/10.1016/j.ab.2011.07.035
- It was also used more recently here: http://dx.doi.org/10.1021/acs.analchem.8b02790

Please note: we initially tried a di-octyl (di-C8) DTPA, and it didn't seem to stably associate with the E. coli. Moving to the di-dodecyl (di-C12) was an effort to match chain length of one leaflet of the outer membrane, and seemed to work well on the bacteria. However, attempts to use the same reagent on human cells weren't as successful. Based on that and some other papers we referenced, I would advise trying to match alkyl chain-length to that of the membrane lipids in your microalgae; for example, if we tried to make a human reagent, we would probably try C14-C18 arms rather than C12.


Mike
Last edited by mleipold on Wed Jul 17, 2019 12:46 pm, edited 1 time in total.
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OLIVIASP

Participant

Posts: 6

Joined: Fri Jun 17, 2016 9:04 am

Post Wed Jul 17, 2019 7:15 am

Re: Cyanobacteria and microalgae

Hi Mike,

Thank you for that value information. We will follow your advise, the research is very interested about do it.

Olivia.

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