[Cytometry] preventing non-specific Ab binding in bovine samples

Mara Rocchi Mara.Rocchi at moredun.ac.uk
Fri Mar 9 05:18:21 EST 2012


Hi Ruth,
I routinely stain bovine/ovine/other animals blood (either whole/lysed or PBMCs) and/or other cells and my background is below 1%. Bovine cells are not too different from human/mouse cells for surface or i.c. staining.

I would suggest checking few points:

How old are the samples?
Have they been in culture?
Has cell viability been checked?
What do the samples look like at the microscope?
Are the cells adherent or in suspension before the staining?
Can the secondary antibody be contaminated/degraded (does she keeps it sterile?),
How old is the secondary antibody?
Is there clumping?
Has the goat serum been heat inactivated?

in addition ABd Serotec now sells a variety of directly conjugated antibodies specific for bovine cell she might want to consider.
If you want to pass her my e-mail I'll be happy to have a chat.

Hope this help
Mara

Mara Rocchi BVM&S PhD MRCVS
Flow Cytometry Manager
Moredun Research Institute
Pentlands Science Park
Bush Loan, Penicuik
EH260PZ
Scotland, UK
e-mail:mara.rocchi at moredun.ac.uk

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-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Ruth Nissly
Sent: 08 March 2012 15:12
To: cytometry at lists.purdue.edu
Subject: [Cytometry] preventing non-specific Ab binding in bovine samples

A cytometrist came to our facility with some very yucky looking results.
She is staining bovine blood cells; she uses primary and secondary antibodies since the commercial availability of conjugated bovine mAbs is pretty slim (and I guess because she does not want to conjugate on her own).
Problem is, if she stains cells with just the secondary antibody, there is lots of fluorescence.  I assume this is due to non-specific binding, and I further assume because the goat-anti-mouse-IgG1 secondary is binding to Fc receptors on the closely-related cow cells (she says she does not see the same issue if she uses a primary-secondary pair with the primary being IgM).
This scientist says she has tried blocking the cells with 5, 10, even 15% goat serum, but the same results.



Does anyone have suggestions or tips from experience with bovine cells?  The obvious simple answer to me is to get a different secondary, but that's not entirely satisfying.



Thanks in advance!



Ruth Nissly



Research Technologist

Microscopy & Cytometry Facility

Huck Institutes of the Life Sciences

Penn State University



W124A Millennium Science Complex

University Park, PA  16802



814-863-2762

 <mailto:rah38 at psu.edu> rah38 at psu.edu



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