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CyTOF to study aged stem cells

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bluesapphire

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Posts: 3

Joined: Thu Apr 07, 2016 11:07 pm

Post Fri Apr 08, 2016 10:52 pm

CyTOF to study aged stem cells

Dear Colleagues:
I’m experiencing some troubles with the Cell Prep and DNA labeling of aged stem cells for CyTOF2 and I hope that you could give me some suggestions with your expertise. I am just starting out using mass cytometry. Right now I’m testing optimal cell Prep conditions to get single cell suspensions. I use DNA intercalator(Ir-191/193) only to look at my cell populations on CyTOF2 during the optimization phase. I have observed that approximately 50% of the events in the fcs file are of low event length and lower Ir -191 intensity than the small percentage of cells labeled with the DNA Ir-191/193 intercalator. I tried different Fix/Perm buffers and different dilutions of DNA intercalator. It looks like The Fix/Perm buffer from ebioscience (used for Foxp3 staining) resulted in more effective labeling of single intact cells than Max par Fix/perm buffer. I also tested 3 dilutions of DNA intercalator, and it seems that 1:2000 dilution works better than 1:1000 and 1:500. There is another problem with these senescent cells. After detaching, 40% cells are dead based on try pan blue staining and create lots of cell debris. I wonder if this can cause lots of non-specific bindings. These aged cells have both DNA condensation and decondensation and I wonder if this kind of cell phenotype may be causing problems with the DNA intercalator? Please review the attached screenshots (dot plots, rain plots and cell suspension images) for more details. The dot plots are DNA (Ir-191) vs. EQ4 beads (Ce140) and DNA (Ir-191) vs. event_length . I’d really appreciate if you could give me some insight on optimizing cell prep, DNA intercalation and/or suggesting alternative strategies to label aged stem cells with a metal-tagged reagent that will aide in cell singlet ID using CyTOF2.
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afs1990

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Posts: 12

Joined: Wed Jul 29, 2015 4:09 pm

Post Wed Apr 13, 2016 3:33 pm

Re: CyTOF to study aged stem cells

Have you tried using a dead cell removal kit?

I've used the Miltenyi dead cell removal kit which did clean up aged blood, but it also removed our monocytes and other phagocytes. I did not do an EDTA wash before using the kit, which may have caused our loss of populations due to platelets binding to those cells. As far as I know, this kit binds to phosphatidylserine on apoptotic cells and activated platelets and removes them.

http://www.miltenyibiotec.com/en/produc ... l-kit.aspx

There is another dead cell removal kit that I found, but I can't find any published data using it.

http://www.biopal.com/viahance.htm

Another idea would be to sort your live cells of interest via FACS first, and then stain for CyTOF.
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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 Wed Apr 13, 2016 7:37 pm

Re: CyTOF to study aged stem cells

Based on how streaky your rain plots are, I think you're still overstaining with Ir.

What is your experimental procedure? Particularly:

1) What buffer do you use during Ir staining?
2) How long do you stain with Ir, and at what temperature?
3) How many washes of what volume do you do after Ir staining?
4) What is the concentration of cells after you dilute to inject on the CyTOF?
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bluesapphire

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Posts: 3

Joined: Thu Apr 07, 2016 11:07 pm

Post Thu Apr 14, 2016 3:06 pm

Re: CyTOF to study aged stem cells

Mike,
Thank you so much for your advice. Here are my answers to your questions:

Because I compared two different Fix/Perm buffer, so I mixed cells with MaxPar Fix/Perm buffer and with Cell-ID Intercalator-Ir as well. For Fix/Perm buffer which I purchased from ebioscience, I washed cells with ebioscience staining buffer twice after Fix/Perm and then mix cells with Cell-ID Intercalator-Ir in PBS. In both methods, I incubated cells with Cell-ID Intercalator-Ir for overnight at 4 degree. After that, all procedures are exactly same as Fulidigm protocol:
* Wash cells by adding 2 ml of MaxPar® Cell Staining Buffer (Fluidigm Cat. 201068), centrifuge and discard supernatant by aspiration.
* Repeat for a total of two washes with MaxPar Cell Staining Buffer.
* Wash cells with 2 ml of MaxPar® Water (Fluidigm Cat. 201069), centrifuge and discard supernatant by aspiration.
* Leave cells pelleted until ready to run on CyTOF. Immediately prior to CyTOF data acquisition, adjust cell concentration to 2.5-5 x 105/ml with MaxPar Water and filter cells into cell strainer cap tubes.

If I have overstained cells with Ir, I'm wondering why the majority cells were not positive at DNA 191 channel? But I twill try more dilutions of DNA intercalator in my next experiments to see if I can get better staining.

Yi
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bluesapphire

Participant

Posts: 3

Joined: Thu Apr 07, 2016 11:07 pm

Post Thu Apr 14, 2016 10:07 pm

Re: CyTOF to study aged stem cells

Hi, afs1990:

Thank you so much for your advice. I'm trying to FASC sorting lice cells before staining and will see if it improves. But the I'm just concerned that one more step of cleaning will loss rare cell population. I will also try this Viahance™ to remove cell debris as well. Thanks so much for your help again.

Yi

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