Staining of C57BL/6 bone marrow cells with the Anti-Mouse Hematopoietic Lineage Biotin Panel (cat. 88-7774) followed by Streptavidin PE (cat. 12-4317) and staining buffer (autofluorescence) (left) or 1.0 µg of Anti-Mouse CD34 Alexa Fluor® 700 (right). Total viable cells were used for analysis.
Contents: Anti-Mouse CD34 Alexa Fluor® 700 Catalog Number: 56-0341 Concentration: 0.2 mg/mL Formulation: aqueous buffer, 0.09% sodium azide, contains stabilizer if necessary Storage Conditions: Store at 2-8°C. Do not freeze. Light sensitive material. Clone: RAM34 Host/Isotype: Rat IgG2a, κ
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56-0341-82
100 ug
Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.
*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.
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.
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. CD34 is a highly glycosylated (approximately 90-120 kDa) member of the sialomucin family and is expressed by capillary endothelial cells, bone marrow stroma, and a small subpopulation of mouse bone marrow cells. RAM34 has been used to purify mouse hematopoietic stem cells (HSC) to near homogeneity. 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 cells that stain with the RAM34 antibody and express undetectable to low levels of the indicated 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
This RAM34 antibody has been tested by flow cytometric analysis of mouse bone marrow cell suspensions. This can be used at less than or equal to 1.0 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 105 to 108 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Note: When staining with the RAM34 antibody an incubation time of 90 minutes is recommended to obtain the optimal signal to noise ratio.
When using direct conjugates of RAM34 to stain mouse bone marrow cells, we routinely perform two-color analysis using RAM34 in combination with a Lineage Cocktail (cat. 88-7772 or cat. 88-7774) to identify lineage-committed bone marrow cells and to better visualize the minor subset of lineage negative/low cells that 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 with two-parameter plots (dot-plots or contour-plots, etc.) for best visualization of the CD34+ population. If you are using only one-color staining, it is recommended to 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. For more detailed information on staining with RAM34, please refer to the following publication PubMed.
References
Palazuelos J, Davoust N, Julien B, Hatterer E, Aguado T, Mechoulam R, Benito C, Romero J, Silva A, Guzmán M, Nataf S, Galve-Roperh I. The CB(2) cannabinoid receptor controls myeloid progenitor trafficking: involvement in the pathogenesis of an animal model of multiple sclerosis. J Biol Chem. 2008 May 9;283(19):13320-9. (RAM34, IHC frozen, PubMed)
Singh P, Yao Y, Weliver A, Broxmeyer HE, Hong SC, Chang CH. Vaccinia virus infection modulates the hematopoietic cell compartments in the bone marrow. Stem Cells.2008 Apr;26(4):1009-16. (RAM34, FC, PubMed)
Lorenz K, Grashoff C, Torka R, Sakai T, Langbein L, Bloch W, Aumailley M, Fässler R. Integrin-linked kinase is required for epidermal and hair follicle morphogenesis. J Cell Biol. 2007 May 7;177(3):501-13 (RAM34, IHC, PubMed)
Iida M, Ihara S, Matsuzaki T. Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles. Dev Growth Differ. 2007 Apr;49(3):185-95. (RAM34, IHC frozen, PubMed)
Park TJ, Boyd K, Curran T. Cardiovascular and craniofacial defects in Crk-null mice. Mol Cell Biol. 2006 Aug;26(16):6272-82. (RAM34, IHC paraffin)
Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996 Jul 12;273(5272):242-5.
