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Quantifying Matrigel in CyTOF

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Jahangir

Contributor

Posts: 36

Joined: Sun Oct 29, 2017 6:34 pm

Post Mon Sep 02, 2019 4:15 pm

Quantifying Matrigel in CyTOF

Hi all!

Hope everyone is well. I've got a question about the above subject.

I'm trying to measure the amount of matrigel in our assay. One way we're thinking of doing this is by chelating an NHS-ester probe to a metal, then binding that probe onto matrigel, wash out the wells a couple times to remove all unbound material, then removing the matrigel from the wells and running that on the CyTOF. My questions are:

1) Is this feasible? i.e. can it work or will there just be huge tyre tracks on the rainplot?
2) Can the above be run in solution mode of the CyTOF? What is the readout of solution mode and how does it actually work? Do you read-out events?
3) Otherwise, if 1) and 2) isn't feasible, does anyone know of any beads which bind to proteins? So we can add those beads into the solution of matrigel and then all of the matrigel would stick to the beads. We would then run those beads on the CyTOF in event mode and we would see the events coming through of those matrigel droplets which have absorbed the NHS-ester probe.

It would be great if anyone has any thoughts on this!

Many thanks,

Jahangir
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jimbomahoney

Master

Posts: 73

Joined: Wed Feb 27, 2019 11:21 am

Post Tue Sep 03, 2019 7:19 am

Re: Quantifying Matrigel in CyTOF

Hi Jahangir,

I can't help on #1, but I (hope) I can explain #2.

Solution mode is like an "average" concentration mode. It's the amount of metal detected over a 1 second period (Tuning mode is the same, but over 4 seconds).

Instead of identifying events (intensities / DCs that meet the threshold criterion and extend for a certain number of 13 µs pushes), solution mode simply gives the total metal in each 1 second "exposure".

I suspect you could use the tuning solution as your reference point - it contains 0.5 ppb of each of the metals (except for Ir, which is 0.25 ppb). In tuning mode (on a Helios), this is generally something like 1.4m DCs for Tb/Tm, or whatever your particular machine tends to tune to. In Solution mode, this would show as ~350k DCs.
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Verhoeff

Participant

Posts: 16

Joined: Wed Mar 02, 2016 10:25 am

Post Tue Sep 03, 2019 8:07 am

Re: Quantifying Matrigel in CyTOF

To expand on James's point slightly, the Transmission Effeciency of the Tuning solution might be different than your metal-NHS ester probe. In the paper on silver nanoparticles (listed on the forum here http://cytoforum.stanford.edu/viewtopic.php?f=10&t=1558 ) they show that the slope of a regression line for dilutions of silver in solution is different than dilutions of NPs. I would suggest making a reference curve by diluting known quantities of the metal-NHS-ester probe.

Jan

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