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Homemade reagents vs fluidigm kit.

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silatour

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Joined: Mon Mar 21, 2022 1:14 pm

Post Mon Mar 21, 2022 3:06 pm

Homemade reagents vs fluidigm kit.

HI CYTOF community,

I started using cytof few month ago and now I’m moving to more « real » experiments involving barcoding and staining of multiple samples.

I have two main questions that are linked :

1) I curently use the Cell-ID Palladium barcoding kit from fluidigm allowing barcoding of up to 20 samples. This kit is insanely expensive. Do you guys usually do your own barcodes or use commercial kit ? And if you use homemade barcode do you usually use the recipe from Zunder and al. ?

2) I usually don’t permeabilize my samples and instead use the fix and perm buffer contained in the kit. When I do my stain, some time antibodies works and some time they don’t work (same antibodies, same type of sample) so I was wondering if permeabilization might be the problem. As it is impossible to know what is in the fix and perm buffer from fluidigm, I was wondering if it could be a transient permeabilization (saponin like) and that when I add my antibodies, depending on how « intact » the cells are sometime the Ab goes in and sometime don’t go in.

Thanks a lot in advances for your help, have a good day.

Simon
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Mar 21, 2022 9:17 pm

Re: Homemade reagents vs fluidigm kit.

Hi Simon,

1. Stanford HIMC uses the Fluidigm Cell-ID barcoding kit when we're barcoding samples that are fixed. Part of it is convenience, and part of it is that we're not set up with lyophilizers ourselves to make the necessary stocks. We do also use antibody-based Live-cell barcoding like CD45, CD298, b2m, or combos thereof; those can also be options for you......for example, CD45 staining on Fixed cells is generally still very good.

2. I'm assuming from your post that your "some time antibodies works and some time they don’t work" is referring to antibodies against intracellular targets.

Back in 2014, Greg Behbehani published a paper ( https://doi.org/10.1002/cyto.a.22573 ) on transient perm of cells to enable small-molecule barcoding prior to surface staining. In that case, the cells were fixed and frozen using SmartTube buffer (probably PROT1), then a transient perm with low concentration saponin (0.02% w/v) was used to perm the cells just enough to get the BC inside but still be pretty rapidly reversible (ie, not letting the intracellular antibodies to stain until a later methanol step).

In most cases, when we use a saponin-based perm agent (like the regular 10X dilution of the eBioscience buffer), we don't know the true final concentration. We do have one protocol where we use purified saponin, and in that case, we use 0.16% w/v final, which is 8-10X that of the transient perm in the Behbehani paper, and is consistent with that paper's statement "The 0.02% saponin concentration is one-tenth that typically used in protocols where saponin is used for intracellular antibody staining."

Some labs have reported issues with Fluidigm Fix/Perm (eg: viewtopic.php?f=3&t=671&p=2043&hilit=fix+perm#p2043 ), though usually more about the "fix" than the "perm".


Mike
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cguidos

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Post Mon Mar 21, 2022 10:13 pm

Re: Homemade reagents vs fluidigm kit.

HI Simon

I think you are asking about 2 different and unrelated topics but the solution may be the same;

1) Issues with Ab staining after using the Fluidigm Barcode kit fix-perm. Fixation in 1.6% FA is well known to compromise some Ab epitopes (I agree with Mike that it is fix and not likely perm that is causing your staining problems). For some Abs fixation mildly decreases staining (sometimes higher Ab concentration is needed for post-fixation staining) but for others there is little or no staining. Potential solutions are finding different clones for the fix sensitive Abs that are fixation-tolerant but this is not always possible. This is why most of us prefer live cell barcoding.

2) Cost of Fluidigm's Pd barcoding kits - you can try home-made fix/perm as per Mike's suggestion but the major cost of the kit is the pre-aliquoted Pd barcodes. Another reason to try live cell barcoding with metal tagged Abs. We tag Abs with isotopically enriched CisPt (4 tags) and then us 2 other metals for 6 tags to give 6 choose 3 = 20 barcodes. We then use DCED-Pd (natural abundance) for live/dead.

Hope that helps
Cindy
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silatour

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Joined: Mon Mar 21, 2022 1:14 pm

Post Mon Mar 21, 2022 10:44 pm

Re: Homemade reagents vs fluidigm kit.

Hi both,

Thank for your quick and detailed response.

I realize that I didn't explain my "problem" properly, sorry

All my staining are intracellular stainings, so I will have to perm at one point. Basically, I was unsure if the barcode perm buffer from fluidigm was transient or if it was sufficient to do intracellular labelling.

Right now my workflow is : fix -> barcode perm buffer->add barcode->wash->add antibodies for intracellular targets.
When barcoding fixed cells and then doing intracellular stainings, what would be your workflow ?

Yeah I looked into the cd45 barcoding kit that look much more "flexible" but unfortunately my cells are not expressing CD45. I didn't know about the B2M version, I'm gonna definitely look into that.

Thanks again.

Simon
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nameerabaig

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Joined: Wed Nov 20, 2019 5:20 pm

Post Mon Mar 21, 2022 11:05 pm

Re: Homemade reagents vs fluidigm kit.

Hello Colleagues,

The fluidigm barcode perm is not sufficient for permeabilizing cells for intracellular targets. We use methanol permeabilization. Additionally, the nuclear perm kit from Fluidigm should be good for intracellular and nuclear targets. The cells should be fixed before permeabilization. You can barcode them first, stain for surface targets, fix cells and then perform nuclear or cytoplasmic permeabilization. Hope this helps.
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BjornZ

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Joined: Fri Jul 10, 2015 1:04 am

Post Tue Mar 22, 2022 1:21 am

Re: Homemade reagents vs fluidigm kit.

As far as barcoding: you can actually use plain Pd nitrate salts with no chelator. This eliminates the high cost of the ITCB and the lyophilizer and saves a ton of time. Last time I ordered the six palladium isotopes, it was $26k for 10 mg of each, $20k of which was Pd-102. If you can find other labs to split an order with and/or only use five of the six isotopes, it quickly pays off vs. the cost of Fluidigm's kit.

The revised barcoding protocol I arrived at is below. I intended to publish this several years ago but never got around to it.

  1. Prepare ~1 × 10^6 cells in a deep-well plate or cluster tube rack, following the layout of the barcode plate.
  2. If volume is greater than 100 µl, centrifuge at 600 x g for 5 minutes, then aspirate, leaving ~100 µl remaining.
  3. Remove protein by washing with PBS: Vortex cells to resuspend pellet, add 1 ml of PBS, centrifuge at 600 x g for 5 minutes, then aspirate to ~100 µl and vortex.
  4. If starting with lysed whole blood or other samples that contain a high amount of serum protein, you may need to repeat step 3.
  5. Add 1 ml of PBS to cells.
  6. Using a 200 µl multichannel pipette, transfer 100 µl of barcoding reagent (see * below) to the cells, mixing thoroughly by pipetting. Repeat for remaining rows.
  7. Centrifuge at 600 x g for 5 minutes, aspirate to 100 µl and vortex.
  8. Add 1 ml of staining buffer to quench the staining. Centrifuge, aspirate and vortex.
  9. Repeat step 8. (This might not be necessary; haven't got to test yet.)
  10. Using a single pipette tip on a single-channel pipette, combine all 20 tubes into one tube. Centrifuge, aspirate and vortex.

* Barcode reagent (Pd(NO3)2) needs to be titrated for your assay (mostly affected by number of cells and how much protein is left after step 3/4), but will likely be close to 30 nM final staining concentration of each isotope (so make a 90 nM stock to make your 3-metal combinations). (May be 500+ nM if lots of cells and/or protein.)

A few things to note:

  • The saponin permeabilization is not necessary. This also eliminates variability due to varying sapogenin content in different lots of saponin.
    pmsaponin.png
    pmsaponin.png (15.01 KiB) Viewed 6532 times
  • There's no timed incubation with the barcode reagent before washing. It's not necessary.
    timecourse.png
    timecourse.png (18.62 KiB) Viewed 6532 times
  • Without the ITCB, there's no chemical prone to hydrolysis. You don't need to rush through this protocol, perform it on ice or keep reagents dessicated at -80 anymore.

Best,
Zach
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mleipold

Guru

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Mar 22, 2022 3:12 pm

Re: Homemade reagents vs fluidigm kit.

Hi Zach,

Thanks for the protocol!

I guess I'm a bit surprised that the Pd would stay stably associated with the cells (ie, not scramble after pooling)....especially since presumably this is just an electrostatic nonspecific interaction, rather than a bonded interaction like is proposed for RuO4 and OsO4.

However, it is consistent with an early 2008 (ICP-MS) paper: https://doi.org/10.1021/ac702128m (see Fig 2 and Fig 3; PdCl2 used)

Is there anything we should know about buffers, like whether Ca2+, Mg2+, or EDTA would affect stability? The 2008 paper did use PBS with Mg2+ and Ca2+, but our typical Rockland PBS doesn't list that ("0X Phosphate Buffered Saline (PBS) consists of 0.2 M Potassium Phosphate, 1.5 M Sodium Chloride, pH 7.2 prepared in highly polished pharmaceutical grade water (WFI)."


Mike
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BjornZ

Contributor

Posts: 43

Joined: Fri Jul 10, 2015 1:04 am

Post Fri Mar 25, 2022 3:23 am

Re: Homemade reagents vs fluidigm kit.

Hi Mike,

Yes, it was rather surprising.

Garry Nolan's lab also uses PBS without Mg/Ca, but I haven't done a stress test to see if there's a way to compete-off the Pd.

Eli Zunder is going to chime in here shortly about plans to address some open questions on the protocol...

Best,
Zach
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GregBehbehani

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Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Mar 25, 2022 5:05 pm

Re: Homemade reagents vs fluidigm kit.

Hi Simon,

I can agree with what has been said by everyone else so far. If you are primarily staining intracellular antigens, the partial-perm barcoding buffer (which is what I think Fluidigm provides with the kit) is unlikely to be good enough to get good staining of the intracellular antigens, and that may be the issue that your are having. The protocol that I published was specifically designed to NOT fully permeabilize the cells, as we would almost always do a methanol perm step before doing the intracellular stain (as Peter Krutzik had long before shown methanol to be a better perm for the things we routinely looked at). We also saw that a full saponin perm could actually strip of some surface antigens (though this was pretty rare). Though it is worth noting that fixed cells (particularly if they were fixed a while ago, or fixed and then frozen) have typically lost enough membrane integrity that the barcoding will work without the additional perm, but it requires more barcoding reagent, and was less consistent in my hands. Also, if you have some cells that were dead at the time fixation, they typically have a membrane that is more permeable and end up with brighter (sometimes too bright) staining. Adding the 0.02% saponin was the minimum amount that consistently eliminated all of this variation, but also did not really allow for full staining of intracellular antigens. What we did not appreciate at the time was how incredibly variable different saponin lots are. Saponin is not in any way a pure substance, and there is very little standardization of the available products. Additionally, some saponin products cause non-specific staining with Fluidigm's newer labeling polymers (which was the source of a product recall several years ago). Fluidigm does now test their saponin lots to make sure that this doesn't happen, but they still don't give very much detail on what exactly is in their buffers, or which polymer is on which antibody that they sell.

I'm not contractually permitted to discuss Fluidigm's barcoding kit, but I can say that I think Zach made some excellent comments on this.

Since you don't need surface marker staining, you may just want to fully permeabilize your cells before barcoding, with whatever perm protocol works best for the antigens you are interested in. Then when you barcode, you can get by with much lower amounts of Pd reagent, and can get 2-3x more uses out of each barcode kit. Like Zach, we also bought our own isotopically purified Pd. If you skip 102, this isn't terribly expensive for 10-population barcoding, but we went ahead and purchased all 6. The cost is pretty high upfront, but you will basically have a lifetime supply of Pd.

Good luck,

Greg
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silatour

Participant

Posts: 6

Joined: Mon Mar 21, 2022 1:14 pm

Post Tue Apr 05, 2022 3:49 pm

Re: Homemade reagents vs fluidigm kit.

Hi all, thanks for all the informations.

As it seems that the ideal permeabilization doesn't exist and that's it's a lot dependent on the antibody targets, I did a try with multiple permeabilization methods.

Here are the results as I guess it might be usefull to share :
Fichier 1-8.png
Fichier 1-8.png (28.03 KiB) Viewed 6136 times


For the markers that i tried, it look like saponin permeabilization after the barcoding is giving the better results.

Simon

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