Compensation slide

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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ssivajothi
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Joined: Thu Jan 11, 2018 9:00 pm

Compensation slide

Post by ssivajothi » Wed Mar 28, 2018 9:02 pm

Hi,

I guess this is a question to the members of the Bodenmiller group: can anyone provide some more information about the preparation of the agarose coated compensation slide?

- Are there any things to keep in mind while preparing it? How thick should the coating be?
- What buffer is the metal solution in before you dilute in Trypan blue? Is there a reason to use trypan specifically?
- How long can we store this slide?
- Storage at RT or 4C?

Any other info is greatly appreciated.

Thank you,
Santhosh
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votti
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Joined: Mon Nov 27, 2017 12:46 pm

Re: Compensation slide

Post by votti » Thu Mar 29, 2018 11:08 am

Hi Santhosh,
Sorry that this took so long! You asked this question already in January and I somehow forgot about it :?

Here is the link to the protocol to generate the spillover slide: https://docs.google.com/document/d/195 ... sp=sharing

To your question:
- coating thickness:
preheating the slide helps to spread the hot agarose evenly (see protocol). As for thickness I just try to cover the slide with the liquid agarose. When it dries the coat gets much thinner.
- I used Trypan blue as this is a metal-free organic dye that we have plentiful in the lab (we get it as a consumable for our cell counter). In the end we just needed something to make the spots visible on the slide. I am happy to hear any other suggestions!
- In the Bodenmiller lab we never saw any indication that storing slides dried at room temperature seems to change the signal in any way. So we store the slide also at RT in a slide box.

Please let me know if you have any suggestions to improve the protocol! I hope it will be helpful.
ssivajothi
Posts: 31
Joined: Thu Jan 11, 2018 9:00 pm

Re: Compensation slide

Post by ssivajothi » Tue Apr 03, 2018 3:57 pm

Hi Vito,

Thank you for posting this. You've done a very good job with this detailed protocol. I will note down any issues/possible improvements as I go through this procedure. Thanks again for sharing.

Best,
Santhosh
ssivajothi
Posts: 31
Joined: Thu Jan 11, 2018 9:00 pm

Re: Compensation slide

Post by ssivajothi » Wed Apr 11, 2018 4:31 pm

Hi Vito,

I am just wondering if the online version of CATALYST is down right now? Is there a special way to access it? Please let me know.

On a separate note, I created the compensation slide using your detailed protocol. I am able to see the Trypan spots pretty clearly. I am just trying to figure out how to perform the compensation in R (I am a little bit "hello world" in that area :D ).

Thanks,
Santhosh
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votti
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Joined: Mon Nov 27, 2017 12:46 pm

Re: Compensation slide

Post by votti » Wed Apr 11, 2018 5:56 pm

Hi Santhosh,

CATALYSTlight (http://imlspenticton.uzh.ch:3838/CATALYSTLite/) seems really to have a severe issues right now. I opened an issue with the developer: https://github.com/HelenaLC/CATALYST/issues/30 and hope that it will be fixed soon.
@Compensation slide: Happy to hear that the protocol was helpful. If you happen to have any comments/suggestions you can also write/comment directly in the Google Docs, then I can improve it over time.

If you have any question about the analysis, there is already a thread in the Data analysis section, where I will be happy to answer.
dlevi
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Joined: Thu May 09, 2019 12:23 pm

Re: Compensation slide

Post by dlevi » Thu May 09, 2019 12:34 pm

Hello all,
I am wondering about the concentration of Fluidigm metals in order to use them for the compensation slide. Does anyone have any information as to how they are supplied and the serial dilutions required for making the slide?
Additionally, the protocol calls for 0.2ul of antibody to be spotted onto the slide, does anyone have any thoughts on pipetting accuracy, and how it can affect the final readout of the slide?
Thank you!
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votti
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Joined: Mon Nov 27, 2017 12:46 pm

Re: Compensation slide

Post by votti » Fri Aug 02, 2019 8:45 am

@Fluidigm stock concentrations: We just back-calculated the concentrations of the stock lanthanides from the antibody conjugation protocol, where Fluidigm writes that the stock solution should be diluted 5ul in 95ul Buffer to get to a 2.5mM final concentration -> 1:20 dilution -> stock has 50mM

@Pipetting accuaracy: The goal is just to get some metal in a sane concentration range (~10-1000 counts per pixel) on the slide while not wasting antibodies. Pitting as little as that does work in our hand to get things in this concentration range and the additional variability due to the pipetting thus should really not matter.
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