FAQ  •  Register  •  Login

Custom barcoding reagents

Forum rules
It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
<<

dahern

Participant

Posts: 16

Joined: Tue Mar 31, 2015 3:45 pm

Post Mon Sep 18, 2017 11:38 am

Custom barcoding reagents

Hi all,

We would like develop our own barcoding reagents as we have found the barcoding kit from fluidigm unsuitable for our needs. We would like to use the CD45 based approach - theres some valuable info in this earlier thread viewtopic.php?f=10&t=262 but maybe things have moved on a little since then with the introduction of the HELIOS and new metal conjugates.

I was wondering if people could share any experience of their own barcoding approach and maybe help us to decide how we go about this. What would the best system need to include - would modelling it on fluidigms triple barcode be the best approach? What are the best mass channels to use for the CD45 barcodes? Is M+16 signal always going to be an issue if we decide to use mass channels within our staining panel range?

Any help much appreciated!

David.
<<

henkmei

Participant

Posts: 18

Joined: Wed Jul 30, 2014 9:44 pm

Post Tue Sep 19, 2017 12:40 pm

Re: Custom barcoding reagents

Hi David,

Besides CD45 Pd conjugates we and others also use Pt and In conjugates; rarely lanthanide conjugates and only if interference via M+1 /b M+16 can be excluded or considered irrelevant. You could also consider using Y89 or Bi209 conjugates along with Pt/Pd/In conjugates for combinatorial approaches. Lanthanide/MAxpar usually provide higher SI compared to In>Pt>Pd. CD45 ab concentrations have to be experimentally optimized. If you do want to use lanthanide/MAXPAR (AM 141-176) conjugates for CD45 Abs I suggest putting all channels next to each other preferably at the high AM end, to avoid interference with analyte specific Abs. You could think of suitable alternatives to CD45 targeting, e.g. if you were to analyze a specific subset only (CD20 barcoding for B cells et c). M+1/M+16 effects of CD45 barcoding channels within your panel range needs to be experimentally addressed. Since CD45 Abs often give high SI as lanthanide conjugates , and can / do spill into +1/+16, I suggest to avoid this. I've just noted a post on spillover correction, that may help in that regard viewtopic.php?f=10&t=826.

Triple labeling is fine and gives you a 20 sample capacity based on 6 channels. You could increase the capacity by using more channels sticking to triple labeling, e.g.8 channels will give you a capacity of 56 samples.  We typically limit experiments / wet work to cell amounts that we can acquire on a single day, usually we are fine with pooling between 3 and 28 samples, and for simplicity we prefer dual labeling which is easier to establish.

Best
Henrik
<<

dahern

Participant

Posts: 16

Joined: Tue Mar 31, 2015 3:45 pm

Post Wed Sep 20, 2017 1:38 pm

Re: Custom barcoding reagents

Thanks Henrik,

I'm thinking we'll go with Pd isotopes. Do you still make the reagents as outlined in your publication or have you made any modifications since that was published?

Regards,

David.

Return to Reagents for CyTOF

Who is online

Users browsing this forum: No registered users and 12 guests