EDTA Concentration during sample acquisition

[Jahangir]

Hi everyone,

I wanted to ask if users add EDTA into the sample for acquisition (if you do so at all)? If so, what concentration do you add? And what is the maximum concentration that can be used which is safe for both the (1) cells and the (2) components of the machine?

We run quite complex samples on the XT (colonic organoid epithelial cells with co-cultures of fibroblasts) and our samples (more often than not) block either the tubing or the nebuliser itself, which causes a lot of stress for both us users and the operators. But adding 2 mM into our sample during our sample acquisition has made the sample introduction much more smooth.

One thing that we tried back in the helios days when the cells were acquired in water was adding 5 mM EDTA into our sample acquisition buffer, but it was stripping off the metals from the antibody. Has anyone ever observed this whilst using a high concentration of EDTA? Also, is there anything in the CAS+ buffer which could affect the activity of EDTA?

Best regards,

Jahangir Sufi

[mleipold]

Hi Jahangir,

Standard Biotools won't tell us what's in any of their buffers. So, there's no way to answer about EDTA and CAS+. You might be able to get answers to specific yes/no composition questions from your FAS, but based on all previous experience, they're not going to give us recipes or full composition listings.

I don't know the upper limit for EDTA concentration in terms of stripping metal. For a decade, we have used CyFACS in most of our staining and wash steps with no (obvious?) effect on metal retention, and that is 2.5mM EDTA. Therefore, if 2mM is improving your runs and 5mM was stripping metal, then I'd probably suggest staying with 2mM. The only impact on machine operation that I could foresee would be slightly increasing total salt concentration and therefore buildup, but if you're running in CAS/CAS+, you're already getting some buildup that you have to periodically clean.

Formally, EDTA would be adding additional carbon to your sample, and carbon-containing buffers aren't recommended for acquisition. However, low millimolar concentration would be the carbon equivalent of maybe a couple hundred cells/sec (see calcs here: viewtopic.php?f=1&t=1405&p=3986&hilit=ammonium+bicarbonate#p3986), which should still be within the max total carbon (cells+buffer) budget for the CyTOF plasma stability.


If Standard Biotools would like to comment further on carbon budget, that would be greatly appreciated.


And thanks for posting about how EDTA improved your acquisition of tissue samples!


Mike

[mleipold]

Hi Jahangir,

A colleague of mine has recently started adding 0.5mM EDTA (about 1/10 what you reported) to her MilliQ samples that are low-quality, especially lower-quality stimmed samples that would be stickier anyway.

She tells me that it's really helped avoid a lot of the clogging she had with 100% MilliQ. The only antibody she has a problem with is 209Bi-CD16 from Fluidigm; in MilliQ-only, it's OK, but in 0.5mM EDTA she sees a lot higher background.


Mike

[mleipold]

A new paper used 5uM EDTA in MilliQ as running buffer: viewtopic.php?f=10&t=3513