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Curiox Laminar wash system

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JamesW

Contributor

Posts: 27

Joined: Tue Nov 21, 2017 6:59 am

Post Mon Nov 25, 2019 9:20 am

Curiox Laminar wash system

Hello all,

I was just wondering if anyone has any experience with the Curiox laminar washers https://www.curiox.com/laminar-wash-system/.

I was interested in them as a way of improving sample quality, staining and cell retention. However, Im a bit concerned that since the maximum volume per well is quite low (70 or 140ul) some of the steps that we normally use larger volumes for, such as Fixation, might prove problematic. Unfortunately Curiox cannot offer a demo in Japan so I cant test it myself.

Best,
James
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JaromirMikes

Participant

Posts: 3

Joined: Tue Sep 29, 2015 7:19 am

Post Tue Nov 26, 2019 1:25 am

Re: Curiox Laminar wash system

Hi James and community.

I don’t have the hands-on experience but we did some development on sample processing automation for CyTOF (even more easily applicable to Flow cytometry):
https://link.springer.com/protocol/10.1 ... 9-9454-0_8

I have to say that I don’t see much of a use (and I apologise to Curiox, but it’s important to sum up some logical assumptions) of this approach to mass cytometry.
1. After all the time spent on sampling, fixation, etc. it doesn’t make much sense to invest in a relatively expensive instrument to do couple of last washes in sake of saving couple of minutes. And you need a centrifuge in your lab anyway.
2. You can’t process high numbers of cells per well…you can of course split larger samples on multiple wells, but that increases cost/sample.
3. If you want to run barcoded samples, you need to process higher number of cells anyway and it’s more logical to do it in a tube….btw, splitting a sample into multiple wells takes time, too.

The main reasons why the cells are lost in washing by centrifugation are following:
1. Decantation instead of aspiration (seriously, people don’t do it - you either have to spin at extremely high g-force or you will loose cells)
2. “Pulse” aspiration with a pipet and not with a constant vacuum suction.
3. Spining in a wrong tube - an angle of the 50mL tube only allows you to suck away down to mark 5mL at the best….you shouldn’t go under that even if you see a nice pellet on the bottom as some cells are spread on walls of the conus in a thin film which is invisible. Transfer cells in a volume of ≈6ml from 50mL to 15mL tube once you're done with large volume processing. The conus angle in 15ml tube is sharper and doesn’t allow the cells to sit there if spun at sufficient g-force. Choosing the right tube is important.
4. Wrong g-force setting either due to a mistake in the protocol or wrong setting on centrifuge as it’s very easy to switch from rpm to g-force setting without noticing it on some models and with a smaller rotors 300rpm is less than 100g (here again, I would like to appeal on major centrifuge producer to do better job on that - use sound, colour or any other indication when the change is done)
5. Multiple washes with pure PBS only - even a little amount of protein covers/blocks tube’s wall and minimises adherence of cells to the wall.
6. Insufficient centrifugation time which significantly depends on centrifuge/rotor vs. liquid column combination. Explanation - the same g-force setting in a tube with a 2cm liquid on the bottom might give different results in comparison to 10cm when tube is full and time is suboptimal especially with a small rotor as it creates a significant gradient of g-force along the tube’s axis as the top of the tube is almost in the centre of the rotor. Larger rotors ensure a bit better conditions. As the result, 3-4 minutes at 300g might be fine when tube is half full but you might need 10min for a full tube in a small centrifuge. Increasing g-force doesn’t speed up much in these situations but increases mechanical damage to cells. Using a larger centrifuge with long radius rotor is a way to get more reproducible results less sensitive to liquid volume. A more detailed protocol giving different centrifugation times for various centrifuges in the lab and various volumes is another way how to solve this.
7. Harsh perm protocols - some cells are lost and there’s nothing to do unless you replace them with gentler ones.

Contamination with RBCs is often caused by improper lysis (irrespective of the lysis protocol) and/or centrifugation at high g-force. Empirically, only one centrifugation at e.g. 800g in first step often seen in many protocols seems to negatively affect contamination with RBCs even if you later wash at lower g-force. 300-400g works fine even for fixed cells in most protocols (even 100g is enough with a pro-longed centrifugation step) and 100-200g is enough for live cells. Above mentioned details related to centrifugation apply.

So if you see loosing a significant number of cells, more than 10% along the whole protocol unless a harsh perm protocol is involved, something is wrong or suboptimal.

To be fair to Curiox’es system, I would see it more suitable in situation where a lot of simple samples needs to be washed after e.g. antibody or fluorescent dyes staining and acquired by flow cytometer. Time-savings in these situations might be significant and worth to consider. High cell loss by “traditional” methods like centrifugation is only a sign of sloppiness and/or bad protocol.

Regards/Jaromir
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charlesm

Participant

Posts: 2

Joined: Thu Oct 17, 2019 2:56 pm

Post Tue Nov 26, 2019 4:15 pm

Re: Curiox Laminar wash system

Dear Jaromir and CyTOF community,

We thank you for your astute analysis on the challenges of sample preparation for mass cytometry! I am Charles Martin from Curiox and would like to address some of these issues you have pointed out.

1. The Curiox Laminar wash system can handle the complete sample preparation including staining, incubation, fixation, and permeabilization as well as the washing steps on a Curiox Laminar Wash 96-well (LW96) plate.

2. One can process from a single cell up to 10 million cells in a single well on the Laminar Wash system. A typical CyTOF workflow will start with PBMCs stained and settled on LW96 plate, followed by subsequent washes, fixation, permeabilization, barcoding and more staining steps, before collecting them for acquisition. Since the washing steps are automated, the Laminar Wash system offers higher consistency and reproducibility even with separately barcoded samples.

In fact, one of the important benefits of the Laminar Wash system is its gentleness on the cells, leading to better viability, debris removal, and separation of the signals even in a harsh condition as pure water. Our CyTOF customers find these benefits valuable especially for samples with low cell numbers.

We invite you to view our CyTOF webinar at our website or to contact us to hear about testimonies and data from our flow and CyTOF users. If your sample size is small, we now offer a 16-well throughput Laminar Wash MINI at an affordable pricing. Thank you!

See my previous posting on a webinar from Linkoping University on sample prep for CyTOF. There are some interesting sample replicate data in this presentation.
viewtopic.php?f=1&t=1638&p=4503&hilit=curiox#p4503
You can watch the webinar here: https://youtu.be/qOP26iVEnQs
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JamesW

Contributor

Posts: 27

Joined: Tue Nov 21, 2017 6:59 am

Post Wed Nov 27, 2019 2:27 am

Re: Curiox Laminar wash system

Dear Jaromir,

Thanks for your advice, actually its partly based on your article that we switched to V-bottom deepwell plates with a fixed height aspirator recently. That was helpful. We don't have a particularly critical problem with cell loss or sample quality but I must admit to always wanting to optimize further.

Dear Charles,

Also thank you, the example data provided is nice.

Best,
James

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