FAQ  •  Register  •  Login

antibody panel interaction

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 :-)
<<

DAVID

Participant

Posts: 17

Joined: Tue Nov 26, 2013 1:55 pm

Post Thu Nov 28, 2013 9:24 am

antibody panel interaction

It appears having a large mixture of Abs in my staining mixture makes some of the individual Abs perform worse (the population separation on flowjo is not as good compared to when there are only 5 or 6 Abs in the staining mix). Has anyone else observed this? Happy to hear your ideas.
<<

Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Thu Nov 28, 2013 11:13 am

Re: antibody panel interaction

In multiplex ELISA such as Luminex this phenomenon is quite common. In Flow this is not observed much, probably due to lower density of antibodies.
Probably a silly question, but did you titrate your antibodies in the same mix (panel) as you use for staining or individually? What titration do you typically use?
<<

mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Dec 03, 2013 8:30 pm

Re: antibody panel interaction

I recommend titering your antibodies as a combined cocktail mixture, *not* as single antibodies. You want to know how they interact *as* a cocktail.

That being said, one of the things about CyTOF staining is a larger number of washes than is typical in fluorescent flow. I think we typically do at least one extra wash at every step than in the corresponding fluorescent protocol. Minimally, any staining step with antibodies gets an extra wash.
<<

DAVID

Participant

Posts: 17

Joined: Tue Nov 26, 2013 1:55 pm

Post Thu Apr 03, 2014 3:49 pm

Re: antibody panel interaction

Thanks for your replies. We usually keep adding a few new Abs to titrate into our current panel (i.e. keep most Ab amounts constant and do a three fold dilution from 3 ug per well to 0.037 ug per well of the new Abs).

Return to Reagents for CyTOF

Who is online

Users browsing this forum: No registered users and 9 guests