Traditional DNA/RNA isolation kits can be used to prepare genomic material for sequencing (RNA-seq, scRNA-seq, WGS, CHIP-seq etc). For some applications with large organoids, tissue specific DNA/RNA extraction kits are recommended. TRI Reagent (T9424) can be added to the dissociated organoids before RNA extraction and can be stored at -80 °C until future use.
SCC111
R-Spondin1 expressing 293T Cell Line
Human
Synonym(s):
RSPO1-expressing 293T cells
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| Pack Size | SKU | Availability | Price |
|---|---|---|---|
| 1 vial | Estimated to ship TODAY | $780.00 |
About This Item
UNSPSC Code:
41106514
NACRES:
NA.81
eCl@ss:
32011203
Biological source:
human
$780.00
Estimated to ship TODAYDetails
Product Name
R-Spondin1 expressing 293T Cell Line, The R-Spondin1-Expressing 293T cell line produces RSPO1, a critical protein used to establish 3D organoid cultures from stomach, small intestine, colon, pancreas, and liver sources.
biological source
human
Quality Segment
shipped in
dry ice
storage temp.
-140 to -196°C
General description
3D organoid culture systems are increasingly employed as powerful tools for the study of human diseases. Organoids are thought to better represent the tissue-specific niche microenvironment. R-spondin-1 (RSPO1) is one of the most extensively used niche factors for culturing 3D organoids. The first published report of an organoid culture system able to promote long-term survival and multilineage differentiation of intestinal stem cells (ISCs) employed mouse Rspo1 as one of the critical factors [1]. Since then, R-Spondin1 has been used to establish organoid cultures from the stomach, small intestine, colon, pancreas and liver from both mouse and human sources [2] [1].
RSPO1 is an intestinal growth factor that works as a potent activator of the Wnt/beta-catenin signaling pathway by binding to leucine-rich repeat–containing G protein–coupled receptors (LGRs), which are expressed in tissue stem cells. RSPO1 also binds to the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3 and triggers their internalization, thereby potentiating Wnt signaling.
The 293T cell line is stably transfected to express mouse R-spondin1 protein tagged with N-terminus HA epitope tag and C-terminus mouse IgG2α Fc region. R-Spondin1 expressing 293T cell line can be used to produce either purified Rspo1 or Rspo1 conditioned media. The FC region and HA tags enable ease of purification and characterization. Cell line is not the published cell line from the Kuo lab, but rather a related cell line developed in the same lab using the reference protocol.
The 293T cell line is stably transfected to express mouse R-spondin1 protein tagged with N-terminus HA epitope tag and C-terminus mouse IgG2α Fc region. R-Spondin1 expressing 293T cell line can be used to produce either purified Rspo1 or Rspo1 conditioned media. The FC region and HA tags enable ease of purification and characterization. Cell line is not the published cell line from the Kuo lab, but rather a related cell line developed in the same lab using the reference protocol.
Application
Research Category
Cell Culture
Cell Culture
Subject to local law, this product is intended to be sold for internal in vitro research use only subject to terms and conditions found here: www.sigmaaldrich.com/restrictedcelluse. This product may not be: re-engineered or copied; used to make derivatives, modifications or functional equivalents; used to obtain patents or other IP claiming use of the product; used to develop, test, or manufacturer a commercial product; used as a component in a commercial product; resold or licensed; used in any clinical applications or trials; or used in humans. A license or limited commercial use agreement is required for use by any for-profit entity, use in services, and use in sponsored academic research. For information regarding any such use, please contact [email protected].
Biochem/physiol Actions
Epithelial Cells
Preparation Note
Store in liquid nitrogen. The cells can be cultured for at least 10 passages after initial thawing without significantly affecting the cell marker expression and functionality.
Analysis Note
• Each vial contains ≥1X10⁶ viable cells.
• Cells are tested negative for HPV-16, HPV-18, Hepatitis A, B, C, Herpesvirus type 6, 7, 8 and HIV-1 & 2 viruses by PCR.
• Cells are negative for mycoplasma contamination.
• Each lot of cells is genotyped by STR analysis to verify the unique identity of the cell line.
• Cells are tested negative for HPV-16, HPV-18, Hepatitis A, B, C, Herpesvirus type 6, 7, 8 and HIV-1 & 2 viruses by PCR.
• Cells are negative for mycoplasma contamination.
• Each lot of cells is genotyped by STR analysis to verify the unique identity of the cell line.
Disclaimer
RESEARCH USE ONLY. This product is regulated in France when intended to be used for scientific purposes, including for import and export activities (Article L 1211-1 paragraph 2 of the Public Health Code). The purchaser (i.e. enduser) is required to obtain an import authorization from the France Ministry of Research referred in the Article L1245-5-1 II. of Public Health Code. By ordering this product, you are confirming that you have obtained the proper import authorization.
1 of 1
This Item | |||
|---|---|---|---|
| biological source human | biological source human | biological source human | biological source mouse |
| shipped in dry ice | shipped in wet ice | shipped in wet ice | shipped in ambient |
| storage temp. -140 to -196°C | storage temp. −20°C | storage temp. −20°C | storage temp. -140 to -196°C |
| Quality Level 100 | Quality Level - | Quality Level - | Quality Level - |
Storage Class
10 - Combustible liquids
flash_point_f
Not applicable
flash_point_c
Not applicable
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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Articles
Organoid culture FAQs. Learn how to culture and analyze organoids following established 3D cell culture protocols. Tips and tricks.
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Akifumi Ootani et al.
Nature medicine, 15(6), 701-706 (2009-04-29)
The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed
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Organoids are primary patient-derived micro tissues grown within a three-dimensional extracellular matrix that better represents in vivo physiology and genetic diversity than existing two-dimensional cell lines. Organoids rely on the self-renewal and differentiation of tissue-resident stem cells that expand in
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Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells
Global Trade Item Number
| SKU | GTIN |
|---|---|
| SCC111 | 04054839226953 |
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How Do you Extract RNA/DNA from Organoids in Cultured in Matrigel?
1 answer-
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How do you stain organoids with antibodies?
1 answer-
Organoids can be stained with antibodies for immunocytochemical (ICC)/Immunohistochemical (IHC) analysis using either whole-mount or paraffin embedding/sectioning techniques. It is critical to use antibodies prequalified for organoid analysis.
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How are organoids cultured?
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Once isolated, organoids are cultured in specialized media embedded in a rich extracellular matrix (ECM) basement membrane extract hydrogel such as Growth Factor Reduced Matrigel. Media is replenished every other day and cells are passaged once per week (7-12 days) before cells become too large or necrotic. Cells can be passaged in either small clump fragments or as single cells using mechanical dissociation/enzyme-free passaging reagents. Depending on organoid type and confluency, split ratios of roughly 1:3-1:4 can be used when passaging organoids. For sensitive organoid types, ROCKi (Y27632) (SCM075) can be added during passaging to help in promoting cell viability.
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What is the selection marker that is used in your RS Spondin expressing 293 cells. Is it neomycin resistance? What antibiotic should be used for selection pressure? Geneticin/G418?
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The cell line is selected by zeocin. It is not necessary to add zeocin to culture medium as the cell line is stable.
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