Flt3 Biotinylated Antibody For FACS

June 06, 2017

BlueskyReddit
Microbiology & Immunology
Columbia University
MD/PhD Student

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Company:

eBioscience™ from Invitrogen Antibodies

Product Name:

CD135 (Flt3) Monoclonal Antibody (A2F10), Biotin

Catalog Number:

13-1351-81

We are interested in the differentiation and self-renewal of progenitors in the hemapoetic system. The Flt3 receptor (CD135) binds Flt3 ligand and is expressed on variety of hematopoetic stem and progenitor cells, particularly of the myeloid branch. We have had difficulty with obtaining good separation of Flt3R+ (CD135) populations using directly conjugated antibodies for FACS. CD135 is an antigen that is not highly expressed, and published FACS analyses showing CD135 expression levels often exhibit poor separation. We selected the Flt3-biotinylated antibody for use with a streptavidin secondary to circumvent this issue and allow for FACS analysis of transcription factor expression by progenitor populations.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

C57BL/6 mouse bone marrow

Primary Incubation

1:100, 30 minutes, 4C

Blocking Agent

FACS buffer (2% FBS in dPBS, 2mM EDTA)

Secondary Incubation

Streptavidin PE-Cy7 (1:400), 20 minutes, 4C

Tertiary Incubation

N/A

Detection

BD Fortessa

Results Summary

Mouse tibia and femurs were flushed to obtain hematopoetic cells which were subsequently stained with a live/dead dye and a cocktail of antibodies to identify progenitor populations. Biotinylated Flt3R antibody was detected using a PE-Cy7 conjugated streptavidin secondary. After gating for live cells, a Flt3R+ population could be identified amongst the lineage negative cells (B220, CD3, CD19, Ter119, Gr1). This population was not visualized when staining with streptavidin secondary only. The positive population was a continuous smear and reached higher MFI intensities than other directly conjugated Flt3R antibodies that we have tried without raising the background fluorescence levels of the negative population.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Reasonable separation of CD135+ cells; low background

The Bad

CD135+ expression is detected as a continuous smear, similar to other antibodies

The Bottom Line

The CD135 biotinylated antibody paired with streptavidin PE-Cy7 provided us with better separation of CD135+ populations than other directly conjugated CD135 antibodies that we've tried, despite the positive signal being continuous with the background, negative population.

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