Product Information

Contents: Alexa Fluor® 700 anti-mouse CD34
Catalog Number: 56-0341
Sizes: 100 ug
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, 0.1% gelatin
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: RAM34
Isotype: Rat IgG2a, κ

Staining of C57Bl/6 bone marrow cells with a cocktail of biotinylated lineage-specific antibodies, followed by streptavidin PE (cat. 12-4317) and staining buffer (autofluorescence) (left) or 1.0 μg of Alexa Fluor® 700 anti-mouse CD34 (RAM34) (right). Total viable cells were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
14-0341	Affinity Purified anti-mouse CD34	N/A	N/A	FC IH/F IHC(Paraffin)
16-0341	Functional Grade* Purified anti-mouse CD34	N/A	N/A	FC
13-0341	Biotin anti-mouse CD34	N/A	N/A	FC
11-0341	Fluorescein isothiocyanate (FITC) anti-mouse CD34	488	518	FC
12-0341	Phycoerythrin (PE) anti-mouse CD34	488	575	FC
17-0341	DISCONTINUED - Please see newly released Alexa Fluor® 647 RAM34 as an alternate Allophycocyanin (APC) anti-mouse CD34	633	660	FC
51-0341	Alexa Fluor® 647 anti-mouse CD34	633	668	FC
56-0341	Alexa Fluor® 700 anti-mouse CD34 (APC-Cy5.5 Equivalent)	633	723	FC
57-0341	Pacific Blue® anti-mouse CD34	402	421	FC
*Functional Grade™ (FG™) Purified: Azide-free, sterile-filtered, and endotoxin < 0.001 ng/µg (unless otherwise noted). *Functional Grade™ (FG™) Biotin: Azide-free, sterile-filtered, and endotoxin < 0.05 ng/µg. Purified: Contains azide, not sterile-filtered, and not endotoxin tested.

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

Description

The RAM34 monoclonal antibody reacts with mouse CD34, also known as mucosialin. It has been reported that the RAM34 antibody can be used to detect CD34⁺Sca-1⁺c-Kit⁺ cells. Upon further optimization, we have determined that the most effective RAM34 formats for this combination of antigen detection are biotin, FITC, and Alexa Fluor 647. Preliminary data suggest that some antibody conjugates (such as PE and APC fluorochromes) may negatively impact detection of CD34. Interestingly, the PE RAM34 conjugate is suitable for the staining of epithelial cells (NIH3T3).

CD34, a highly glycosylated (approximately 90-120 kDa) member of the sialomucin family, is expressed by capillary endothelial cells, BM stoma and a small subpopulation of mouse bone marrow cells. RAM34 has been used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the CD34 low to negative fraction. CD34 expressed on endothelial cells is a ligand for CD62L and plays a role in adhesion. Simultaneous staining of mouse bone marrow cells with a cocktail of antibodies to lineage markers (CD3, CD11b, Ly6G, TER-119 and CD45R/B220) reveals a subset of total BM cells staining with RAM34 that can express undetectable to low levels of the mentioned lineage markers.

Applications Reported

For research use only, not for diagnostic or therapeutic use. This RAM34 antibody has been reported for use in flow cytometric analysis.

The Alexa Fluor® 700 emits at 723 nm and can be excited with the He-Ne 633 laser. Most instruments will require a 685 LP mirror and 710/20 filter. Please make sure that your instrument is capable of detecting this fluorochome.

Applications Tested

The RAM34 antibody is routinely tested by flow cytometric analysis of a mouse cell line (NIH/3T3) and mouse bone marrow (BM) cell suspensions. When using conjugates of RAM34 for staining of mouse BM cells, we routinely perform a two-color analysis using RAM34 in combination with Lineage Cocktail (cat. 88-7774) to identify lineage-committed BM cells and better visualize the minor subset of Lineage negative/low cells that normally stain with RAM34 as reported in the literature. Gating strategies that exclude cells with low level expression of lineage markers may significantly decrease the total number of RAM34-positive cells. If using a Lineage Cocktail and/or other markers such as CD117/c-Kit or Ly6AE/Sca-1, it is best to analyze data on two-color plots (dot-plots or contour-plots, etc.) for best visualization of the staining. If you are using only one-color staining, analyze data as a two-parameter plot of RAM34 staining vs. Forward Light Scatter (FSC). Collecting and analyzing >10,000 total events per sample is helpful in increasing the number of RAM34-positive cells.

It is recommended to use 1 µg per million BM cells in a 100 µl total staining volume. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. For more detailed information on staining with RAM34, please reference the following publication PubMed.

References

Osawa M, Hanada K, Hamada H, Nakauchi H. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 273(5272):242-5.

Related Products

Cat. 56-4321 Alexa Fluor® 700 Rat IgG2a Isotype Control (APC-Cy5.5 Equivalent)

Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. This product is subject to an agreement between Molecular Probes, Inc. and eBioscience, and the manufacture, use, sale or import of this product may be subject to one or more U.S. patents, pending applications and corresponding foreign equivalents, owned by Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corp). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product for life science research or as an ASR. The buyer cannot use this product for manufacturing or for any other screening (specifically including use in combination with microarrays or High Content Screening) or testing purpose, other than as an ASR. For information on purchasing a license to this product for purposes other than life science research or use as an ASR, contact Molecular Probes, Inc.