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Tubes for storage

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DMcDonald

Contributor

Posts: 21

Joined: Thu Nov 26, 2015 10:14 am

Post Fri Jan 15, 2021 11:57 am

Tubes for storage

Hi All,

I appreciate this isn't the sexiest of subjects but what tubes do you store you self-conjugated Abs in?

We're about to conjugate up some new panels and are considering using 0.2mL matrix tubes in latch racks as these are fantastic for large genomic cytometry panels. We've had issues in the past with some low volume, screw top tubes having various metal contaminants in the detector range so any recommendations or warnings (particularly on the above) would be much appreciated.

Thanks in advance,
David
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jan 15, 2021 6:25 pm

Re: Tubes for storage

Hi David,

For the master stocks after doing a 100ug conjugation, I use a 1.5mL microcentrifuge tube. That's convenient, as I have a ton of them and the volume after stabilizer is in the 100-200uL range. I haven't had any contamination issues, though I do steer to the clear/colorless ones, as sometimes the colored ones do have metal providing the color......

I usually do my single-sample staining in 50uL cocktail. If I make single-use aliquots for frozen cocktails, then I use 0.2mL PCR tubes with the flip-cap (no o-ring, etc). Basically, the size and shape of the X8 polymer tubes (just usually in single-tubes in strip-tubes).

I think the screw-cap contaminants that you mention are probably coming from the o-ring: sometimes rubber either has a contaminant left over from the polymerization (ie, probably impregnated throughout the rubber), or is something adsorbed to the rubber during manufacturing (ie, on the surface). You might have seen this over the years when you change an o-ring on the CyTOF, and occasionally seen a spike in lead, iodine, etc that eventually goes away as you run Wash or Tuning Solution (acid stripping it out).

I've never used either of these 0.6mL microcentrifuge tubes, but they do state that they're "metal-free":
https://www.thomassci.com/Laboratory-Su ... ee%20Tubes
https://www.thomassci.com/Laboratory-Su ... ee%20Tubes


You might also try reaching out to a chemistry department or earth science department at your university: someone must be doing metal analysis, and might be able to recommend a tube.


Mike
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DMcDonald

Contributor

Posts: 21

Joined: Thu Nov 26, 2015 10:14 am

Post Mon Feb 01, 2021 10:34 am

Re: Tubes for storage

Hi Mike,

Thanks for the reply. So we tested the matrix latch racks in both a 200uL and 1mL format and they were nice and clean after leaving clean water in them for a week. They're proving really handy for keeping things together and large panels can be spun down quickly on a plate rotor. We're using the 1mL format tube for stocks but we may consider freezing cocktails in the 200uL format. In your experience of this have you had any negative experience or caveats to freezing the master mixes?

Best wishes,
David
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Feb 01, 2021 3:55 pm

Re: Tubes for storage

Hi David,

So far, I haven't had any antibodies "fail" after freezing master mixes. I dilute my cocktail like normal (ie, 50uL staining volume), then freeze in single-use aliquots.

I haven't tried re-freezing; I'm hesitant to try that from an antibody point of view, which is why I make single-use (1rxn) aliquots. We are in a study with another group who always have the same number of samples (20 per plate), so they made 20rxn aliquots.

While it hasn't been an issue for us so far, I might also recommend that you keep your cocktails in a freeze that is *not* frost-free (ie, you have to scrape it). Most -80C freezer are that way anyway, but just in case yours isn't, the temp cycling for frost-free freezers can add to normal sublimation issues with the water in the cocktail. I think we've all had cases (eg, Ir 2000x stock) where we go to the freezer and there's only a solid residue at the bottom and a bunch of ice crystals at the top. For something like Ir that's a small molecule, it's not a big deal to thaw and redissolve, but biomolecules don't often like being (effectively) lyophilized without some sort of cryoprotectant.


Mike
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DanielBachurski

Participant

Posts: 11

Joined: Sun Jul 05, 2020 4:10 pm

Post Fri Mar 12, 2021 8:59 pm

Re: Tubes for storage

Hi Mike,

Just a few questions concerning your single-use AB cocktails, if I may. Do you use cell staining buffer/permeabilization buffer to dilute the antibodies to 50 µL before freezing at -80°C, or do you use antibody stabilizer (Candor Bioscience)? Do you centrifuge your aliquots directly before use (12k rcf 5 min)? Are you combining lanthanide- and cadmium-conjugated antibodies in one aliquot?

Thank you so much!
Best
Daniel
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Mar 15, 2021 2:54 pm

Re: Tubes for storage

Hi Daniel,

I use our CyFACS (1x PBS+BSA+EDTA) to dilute to working concentration, then spin filter, then aliquot and freeze at -80C. I don't use Candor buffer to dilute. I also don't re-centrifuge the aliquots after thawing, but I can't imagine it'd be a problem if you did.

We have not tried combining Cd MCP9 antibodies with Ln X8/DN3 antibodies. We've successfully frozen them separately (as surface cocktail for Ln and BC cocktail for Cd), just haven't had a reason to mix them yet.


Mike
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Mon Mar 15, 2021 8:30 pm

Re: Tubes for storage

Hi Mike et al

Wondering if anyone has tried freezing cocktails at 2X-5X? We'd like to freeze more concentrated stocks to minimize storage volume and to allow the option to mix different add-on Abs into a pre-frozen base cocktail.

Cynthia
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Mar 15, 2021 8:56 pm

Re: Tubes for storage

Hi Cynthia,

We're in the middle of a large study where the batches are 4 plates of 20 samples, with each plate separately barcoded. Each frozen aliquot is enough for all 80 samples, rather than 80 individual frozen aliquots or even 4 individual frozen aliquots each serving 20. This gives less flexibility for the few batches which have fewer than 80 samples or 4 plates (ie, you're thawing enough for 4x20 regardless), but means you have 1 tube per batch to thaw, rather than 80 or 4.

The group who made the aliquots did make them in 100uL which we then thaw and dilute to 400uL, spin filter, then split 4 x 100uL. So, technically, I guess that means that they're 4x conc.

In this case, I think this was more a logistical thing than anything, since the tubes they used only hold 200-300uL rather than 400uL. In your case, for maximum flexibility (variable sample number), I'm not sure how many tests you would be trying to pack into 1 tube (which would help decide how concentrated you would want to make it to fit into a 200-500uL tube).


Mike
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DanielBachurski

Participant

Posts: 11

Joined: Sun Jul 05, 2020 4:10 pm

Post Thu Mar 18, 2021 9:10 pm

Re: Tubes for storage

Hi Mike,

Thank you for sharing your experience. This is an incredible help.

I am always a bit concerned about losing antibodies due to protein binding to the filter unit. Which filter do you use to concentrate and buffer exchange your Ln antibody master mixes before aliquoting (Amicon ultra 50k)?
Could you maybe describe in detail how you spin filter the ABs before aliquoting?

Daniel
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Mar 18, 2021 9:17 pm

Re: Tubes for storage

Hi Daniel,

I use Millipore Ultrafree MC VV Durapore PVDF 0.1um (Ref UFC30VV00). I think they hold about 500uL of volume with no problem.

I generally assume that the filter will "eat" 1 test of antibody cocktail, due to membrane wetting, etc. We're now doing a project with a lab who pre-wets their filter (add buffer, spin at 12-13K xg); I haven't exhaustively tested whether this improves recovery.


In short: I make however much 1x cocktail I'm going to need, spin through the Durapore filter (or filters, if necessary) usually 12-13K x g, then aliquot into the single-use PCR tubes and freeze at -80C.


Mike
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