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Fluidigm protocols?

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ssivajothi

Contributor

Posts: 29

Joined: Mon Aug 01, 2016 2:31 pm

Post Fri Oct 21, 2016 8:16 pm

Fluidigm protocols?

Hi all,

I am new to the Cytof world, it is nice to have an informative and supportive community here. I will be managing a Helios instrument at JAX-GM in Farmington, CT. I am currently standardizing procedures for getting the instrument and service running and I have a couple of newbie questions:

1. How have the Fluidigm protocols worked for everybody? I have seen other protocols with custom buffers; is that purely a matter of cost reduction?

2. What is your general workflow for testing a new Ab? Buy pure--use fluorochrome secondary for flow testing-- metal conjugate?Also, has anyone encountered issues with metal conjugating polyclonal antibodies from rabbit?

3. Have you been able to successfully combine intracellular and nuclear antigen staining into the same procedure?

Thank you in advance for any input.

Sincerely,
Santhosh Sivajothi
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 24, 2016 9:27 pm

Re: Fluidigm protocols?

Hi Santhosh,

1. I have only tested the Maxpar Cell Surface Staining protocol (PRD012), as most of my work focuses on surface phenotyping of live cells. In general, the protocol worked well. However, I would advise you to test them out against regular protocols to make sure that everything is working consistently, as well as check titrations and other things.

For example, I tried 20 antibodies from Fluidigm's catalog (the 17 marker surface phenotyping kit, plus 3 more antibodies). I think 12 of them, I used at 1uL/test. Of the remainder, about half were at 0.5uL/test and half at 1.25uL/test. I also know that the same conjugate used under different staining conditions might have different titers: for surface phenotyping, we use the CD45 conjugate at 0.8-1uL/test, but for some phospho assays, we use it at closer to 0.2-0.25uL/test or we get a tire track in the rain plot. When I use cisplatin as a live-dead, I typically use less than the recommended concentration and stain for 5 min rather than 1 min, so I can increase my reproducibility on an entire plate of samples.

Similarly, I have heard of 1-2 people having an issue with some specific surface markers when using Fix/perm buffer; the only way to find out is to test it vs PFA+saponin or whatever.


Regarding buffers and cost reduction: at the HIMC, we developed many of our staining protocols before Fluidigm started selling pre-conjugated antibodies, let alone buffer kits. We didn't want to go back and rework our protocols just to accommodate Fluidigm buffers unless we had a reason to, especially when the in-house buffer recipes are only PBS+BSA (+-EDTA).


2. When we test out a new antibody, we typically look at what is available in the catalog from major suppliers like BD, Biolegend, R&D Systems, etc, and try to pick the one with the brightest signal. Of course, that's assuming there isn't clone-specific published or known about how well something works under specific assay conditions (eg, post-fix, post-methanol, etc). We usually try testing out primary MAXPAR conjugates, and only resort to secondaries when the primary may not be working for a really desirable clone.

I haven't tried polyclonal rabbit antibodies, so I can't comment.


Mike
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ssivajothi

Contributor

Posts: 29

Joined: Mon Aug 01, 2016 2:31 pm

Post Wed Oct 26, 2016 6:40 pm

Re: Fluidigm protocols?

Hi Mike,

Thank you so much for the detailed reply. I will be mindful of your recommendations. I will stick with Fluidigm kit/reagents for now and test custom buffers/perms when I get a chance. I will be trying conjugations on affinity purified polyclonal rabbit antibodies. I will update with the results.

Just another question: Do you make determinations of 'metal tag to antigen' matching (for panel design) just based on the density of these antigens as reported in literature or do you perform some other tests to determine the best channel for a particular antibody?

Thank you,
Santhosh

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