Anti CD44v10-e16 - Cosmo Bio Co.,Ltd.

Antibodies

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Anti CD44v10-e16 Antibody

Catalog No.: CAC-LKG-M002
Size: 100UG
Price: ¥100000
$1334
antigen/source: CD44v10-e16
host: Rat
Description: Mouse CD44v8-10 transfected cell
Label: Unlabeled
Purity: Ig fraction - Protein G
Application: Flow Cytometry
Storage: 4C
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Immunogen: Mouse
Isotype: IgG2a
Clone: RM1
Reacts with: Mouse

Other:

 Background 
CD44 is a single-pass type I transmembrane protein and functions as a cellular adhesion molecule for hyaluronic acid, a
major component of the extracellular matrix. It exists in numerous isoforms that are generated through alternative splicing of CD44 precursor mRNA. Whereas the standard isoform of CD44 (CD44 s) is expressed predominantly in hematopoietic
cells and normal epithelial cell subsets, CD44v (variant) isoforms, which contain additional insertions in the membrane-proximal extracellular region, are highly expressed in epithelial-type carcinomas). Moreover, CD44 is reported as cell surface marker for cancer stem cells (CSCs) derived from solid tumors including breast, prostate, colon,
head and neck and pancreatic cancer. Expression of CD44, especially variant isoforms (CD44 v8-10), contributes to reactive oxygen species (ROS) defense through upregulation of the synthesis of reduced glutathione (GSH), the primary intracellular antioxidant. CD44 v8-10 interacts with and stabilizes xCT, a subunit of the cystine-glutamate transporter xc(-), and thereby promotes cystine uptake for GSH synthesis. The ability to avoid the consequences of exposure to high levels of ROS is required for cancer cell survival andpropagation in vivo. CSCs, in which defense against ROS is enhanced by CD44v8-10 are thus thought to drive tumor growth, chemoresistance and metastasis). Clone RM1, is a monoclonal antibody specific for mouse CD44 v10-e16 can be used for FCM assay,and importantly, for the enrichment of CSCs using a cell sorter. RM1 can be applied towards understanding a variety of molecular mechanisms for cancer stem cells using in vitro cell-based assays such as gin vitro sphere formation assayh and gin vivolung metastasis assay".
 Protocol  Flow cytometry protocol (Cell Analysis)
A. Cell Preparation
1. Remove cells from incubator.
2. Discard culture medium.
3. Briefly rinse the cell layer with PBS.
4. Add 0.25% trypsin-EDTA solution to dish.Return the dish to the incubator and incubate for 2-10 minutes or until cells are detached.
5. Resuspend cells in complete growth medium to inactivate the trypsin.
B. Staining
1. Aliquot 1x105cells into each assay tube.
2. Add 150 l0.2 %BSA in PBS to each tube and rinse by centrifugation.
3. Add 50 l diluted primary antibody (3 g/ml RV3 in 0.2 % BSA in PBS) to the assay tubes.
4. Incubate 45 minutes at 4 C.
5. Add 100 l 0.1 % BSA in PBS to each tube and wash by centrifugation.
6. Wash two times in 150 l 0.1 % BSA in PBS by centrifugation.
7. Resuspend cells in 50 l PE-labeled secondary antibody solution (Jackson Immuno Research 712-116-153),diluted 1:200 in 0.1% BSA in PBS.
8. Incubate 30 minutes at 4 Cin the dark.
9. Add 100 l 0.1 % BSA in PBS to each tube and wash by centrifugation.
10. Wash two times in 150 l 0.1 % BSA in PBS by centrifugation.
11. Resuspend cells in 100l PBS.
12. Add 100l Propidium Iodide (SIGMA, P4864), diluted 1:500 inPBS, to stain dead cells.
13. Analyze using flow cytometry.
 Protocol  Flow cytometry protocol (Cell Sorting)
A. Cell Preparation
1. Prepare cultured cells for sorting based on the ratio of the CD44v expression cells.
2. Remove cellsfrom incubator.
3. Discard culture medium.
4. Briefly rinse the cell layer with PBS.
5. Add 0.25% trypsin-EDTA solution to dish. Return the dish to the incubator and incubate for 2-10 minutes or until cells are detached.
6. Resuspend the cellsincompletegrowth medium to inactivate the trypsin.
B. Staining (for 1x107 cells)
1. Aliquot 1x107 cells into 15ml tube.
2. Add 10 ml 0.2 % BSA in PBS to the tube and rinse by centrifugation.
3. Add 5ml diluted primary antibody (3 g/mlRV3 in 0.2% BSA in PBS) to the tube.
4. Incubate with gentle agitation 45 minutes at 4 C.
5. Wash three times in 10 ml 0.1 % BSA in PBS by centrifugation.
6. Resuspend cells in 5 ml PE-labeled secondary antibody solution (Jackson Immuno Research 712-116-153), diluted 1:200 in 0.1 % BSA in PBS.
7. Incubate 45 minutes at 4 C in the dark.
8. Wash three times in 10 ml 0.1 % BSA in PBS by centrifugation.
9. Resuspend cells in 5 ml PBS.
10. Add 5ml Propidium Iodide (SIGMA, P4864) diluted1:500 in PBS, to stain dead cells.
11. Sort CD44v high and low expression cells using a cell sorter.
12. Wash the sorted cells in 5 ml complete growth medium (added antibiotic drug) three times by centrifugation.
13. Culture the sorted cells andscale up.
ENote the passage number and analyze the cell population periodically using flow cytometry.
14. If desired, sort the cells again, they would be high-enrichment.



 IMAGE1



Flow cytometry analysis of CD44 v in Mouse Breast cancer cell line 4T1 with anti-CD44 v10-e16 (RM1, 3g/mL) antibody and PE-labeled anti Rat IgG antibody.

 IMAGE2



Flow cytometry Cell Sorting of CD44 v expression level in Mouse Breast cancer cell line 4T1 with anti-CD44 v10-e16 (RM1) antibody and PE-labeled anti Rat IgG antibody. Two kinds of subpopulations gCD44v+(High) and CD44v-(Low)h were isolated.

Reference:

•  Nagano O., et al.,, Oncogene. 2013 Jan 21., 1-8. PMID:23334333
•  Ishimoto T., et al., Cancer Cell. 2011 Mar 8;19(3):387-400. PMID : 21397861
•  Yae T., et al.,, Nat Commun. 2012 Jun 6;3:883. PMID: 22673910
•  Tsugawa H., et al.,, Cell Host Microbe. 2012 Dec 13;12(6):764-77. PMID: 23245321
•  Tanabe KK., et al.,, Lancet. 1993 Mar 20;341(8847):725-6. PMID: 8095628
 
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