CD45-Cd Titration

[EftyCh17]

Hello all,

I am planning of using the Cd-CD45 barcodes to pool together three different samples for the MDIPA staining. Could you let me know any tips or things to consider before starting?

Do I need to titrate the barcodes? Is it better to titrate the combinations together or one-by-one?

Thanks a lot.

[mleipold]

Hi Eftychia,

I would recommend that you titer them as you plan to use them in the experiment.

So, if you're doing combos (eg, trios like 6-choose-3), then make the mixture and use the mixture for your titration.


Note: this is assuming that the SBio-conjugated Cd-CD45 commercial conjugates have at least roughly similar staining intensity (eg, 1uL would give you say 1e2 signal in each of the BCs you test). I haven't used them: I made my own before SBio started selling the pre-conjugated, so I can't formally answer that. But, that's something you can check when you inspect your results. Or, do that as a first experiment just to reassure yourself that each channel is behaving similarly.


One question: you say you're going to "pool together three different samples for the MDIPA staining." So, are you using a 3-choose-1 scheme, where each of the three different samples would receive a different single Cd-CD45?


Finally: remember, the CD45 BCs use the same HI30 clone as the MDIPA 89Y-CD45. So, if you're going to barcode each of your 3 samples, then pool them, *then* stain with MDIPA, you should expect that your 89Y-CD45 signal will be lower than it would be without the CD45 BC.


Mike

[EftyCh17]

Hello Mike,

Thank you for your reply.

So, would you say it is best to titrate and also stain for the MDIPA pellet?

In regards to your question, I will barcode each sample with 3 different Cd-CD45 and then pool together all three.

About your final note, do you think its better to stain with only two Cd-CD45 for each sample? Or can I use the CD45 BC present only to one of the samples to gate the different CD45+ populations?

Thanks a lot
Eftychia

[mleipold]

Hi Eftychia,

I generally recommend that you do all development work (titrations and such) as you would perform them in your planned experiment, whenever possible.

In particular, cell number/count is an important factor in all titrations and staining steps. Here's the MDIPA staining protocol: https://fluidigm.my.salesforce.com/sfc/ ... Gkpahnsipk

It's geared to 270uL of WB or 3M PBMCs. So, if the size of your *total pool* differs substantially from those parameters, you may need to increase or decrease the amount of MDIPA used for your pool. In other words, 0.5M PBMCs or >5M PBMCs may need to be retitered.

For example: in some of my experiments (my own in-house reagents), I would stain individual samples with a given amount of antibody cocktail (ie, 1 sample = 1M PBMC stained with 1 test of the cocktail). However, when I did barcoding and was pooling 10 samples, I did *NOT* usually need the same ratio (Ie, I did *not* need 10 tests of cocktail for 10M PBMCs)....in most cases, I needed less cocktail. This likely meant that I was slightly overstaining my individual samples, which when multiplied meant that I was substantially overstaining the pool (and causing a lot of streaking and background).


Regarding CD45 BC and 89Y-CD45 in MDIPA: you'll have to test this in your hands. This may result in only a slight decrease in 89Y-CD45 signal, but could be significant. You can formally make the assumption that anything that makes it through debarcoding is CD45+, since you're using CD45 BC. But you probably want to officially state that in your protocol and analysis.


Re number of samples, number of BC channels: if you only have 3 samples in each pool, I would personally suggest just using a single BC channel per sample (so, 3-choose-1, total of 3 BC channels). Since each sample only has a single BC, there's no decrease in signal due to antibody competition of different channels within the same BC (discussed a bit more here: viewtopic.php?f=1&t=5263). There's also the practical benefit of not having to mix the different BC combos yourself.


Mike