R spondin

Manufactured by Thermo Fisher Scientific

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R-spondin is a recombinant protein that acts as an agonist of the Wnt/β-catenin signaling pathway. It is primarily used in cell culture applications to promote the expansion and maintenance of stem cells and other cell types.

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R-spondin-1 (52 citations) Recombinant human R-spondin 1 (17 citations) Human R-spondin-1 (8 citations) Murine R-spondin-1 (3 citations) R-Spondin CM (2 citations) R-Spondin 3 (2 citations)

34 protocols using r spondin

Intestinal Crypt and Myofibroblast Co-Culture

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Myofibroblasts and crypts were isolated as stated above from 6-week–8-month-old mice (n = 3 for the 2-day cultures and n = 4 for the 7-day cultures), expanded, and then used for the experiment. Acellular and cellular matrix were prepared as previously published [14 (link)], with minor modifications. 900 μL of acellular matrix was pipetted on a transwell (24 mm transwell with 3.0 μm pore polycarbonate membrane insert, Corning), which was placed into a 6-well transwell carrier (Organogenesis). After the solidification, 450 μL of cellular matrix containing intestinal myofibroblasts was poured. After the stabilization of the matrix, 450 μL of cellular matrix containing intestinal crypts was poured on the top. Per well 7.5 mL medium containing RPMI1640 (Life Technologies), 10% FBS (Life Technologies), 1% Penicillin/Streptomycin (Life Technologies), and 330 ng/mL R-Spondin (Peprotech) were added. At day 0 cells were seeded. R-Spondin was added to the medium only at day 0. At days 2 and 7 the culture was fixed in formalin, dehydrated, and embedded in paraffin. On the cut paraffin slides H&E, Ki-67 (Abcam), and α-SMA (Abcam) staining were performed. Total number of crypts was quantified on slides that were stained for H&E. Disintegrated crypts were counted as nonviable. Survival was defined as percentage of viable crypts.

Pastuła A., Middelhoff M., Brandtner A., Tobiasch M., Höhl B., Nuber A.H., Demir I.E., Neupert S., Kollmann P., Mazzuoli-Weber G, & Quante M. (2015). Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche. Stem Cells International, 2016, 3710836.

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Effects of RCBB on Intestinal Stem Cells

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We then investigated the effects of the RCBB chemical cocktail on the proliferation and differentiation of mouse ISC. Proximal small intestinal crypts were isolated from adult C57BL/6J mice as previously described (8 (link)). Isolated crypts were embedded in Matrigel (growth factor reduced, Corning). The basal DMEM/F12 medium supplemented with 1% N2, 1% B27, 1 mM N-acetylcysteine (MedChem Express), and 1% P/S was added, containing the ENR growth factors including EGF (50 ng/ml; PeproTech), Noggin (100 ng/ml; PeproTech), and R-spondin 1 (500 ng/ml; PeproTech). To investigate the effects of the RCBB cocktail on the crypts, passaged intestinal crypts were first cultured under ENR condition for 4 days and then replaced with ENR + RCBB. After 4 days of chemical induction, crypts were fixed, and fixed cells were used for alkaline phosphatase assay and PAS staining and immunostained for the intestinal cell markers CYP3A4, LYZ, and ChrA and the proliferation marker Ki67. The images were taken by a light microscope and a confocal microscope.

Zhao A., Qin H., Sun M., Tang M., Mei J., Ma K, & Fu X. (2021). Chemical conversion of human epidermal stem cells into intestinal goblet cells for modeling mucus-microbe interaction and therapy. Science Advances, 7(16), eabb2213.

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Intestinal Organoid Assay with PA Treatment

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Organoid assays were performed as described previously^{81 (link)}. Briefly, human intestinal normal epithelial cells were maintained with organoid culture advanced DMEM/F12 containing growth factors (100 ng/mL Noggin (Peprotech), 500 ng/mL R-spondin (Peprotech), 50 ng/mL epidermal growth factor (Peprotech) and 10 μM Y-27632 (Abmole)) and treated with PA (300 μM). After spheroid organoid formation for 5 days, organoids were photographed and measured in diameter.

Zhang Q., Yang X., Wu J., Ye S., Gong J., Cheng W.M., Luo Z., Yu J., Liu Y., Zeng W., Liu C., Xiong Z., Chen Y., He Z, & Lan P. (2023). Reprogramming of palmitic acid induced by dephosphorylation of ACOX1 promotes β-catenin palmitoylation to drive colorectal cancer progression. Cell Discovery, 9, 26.

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Crypt Isolation and Organoid Culture

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The intestines were opened longitudinally, and villi were scraped away. The pieces were thorough washed in cold PBS, and incubated in 2 mM EDTA solution in PBS for 10 min at 4 °C. Then, EDTA solution was replaced with PBS and shacked vigorously for 45 s. Crypt fractions were purified by successive centrifugation steps. For every 500-1,000 crypts, a mixture of 100 μL Matrigel (BD Biosciences) and complete growth medium (ratio 2: 1) is added. After polymerization, 100 μl Advanced DMEM/F12 (Invitrogen) containing EGF (50 ng/ml, PeproTech), R-spondin (500 ng/ml, PeproTech), Noggin (100 ng/ml, PeproTech), N-acetyl-1-cysteine (1 μM, Sigma-Aldrich), N2 (1X, Life Technologies) and B27 (1X, Life Technologies) was added and refreshed every two or three days. On the fifth day, the organoids were stained with 7-AAD (Invitrogen, A1310) for 5 min to detect apoptosis levels, and photos were imaged by Zeiss fluorescence microscope and quantified by Image J software. (The NAC concentration in organoid culture is extremely low (1 μM), which will not affect the level of ROS.)

Liu M., Rao H., Liu J., Li X., Feng W., Gui L., Tang H., Xu J., Gao W.Q, & Li L. (2021). The histone methyltransferase SETD2 modulates oxidative stress to attenuate experimental colitis. Redox Biology, 43, 102004.

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Induction of TIGAR in Organoids

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Isolated crypts were suspended with Matrigel (Corning, Cat#356231, growth factor reduced), which was overlaid by the organoid medium. Advanced DMEM/F12 (Thermo Fisher Scientific, Cat#12634-010) medium was supplemented with EGF 40 ng/ml (Peprotech, Cat#315-09-100), Noggin 100 ng/ml (Peprotech, Cat#250-38-5), R-spondin 500 ng/ml (Peprotech, Cat#315-32-5), N2 1× (Thermo Fisher Scientific, Cat#17502048), B27 1× (Thermo Fisher Scientific, Cat#17504044), and Y-27632 dihydrochloride monohydrate 10 μM (Sigma, Cat#Y0503). Intestinal crypts were cultured in the above mentioned media in Matrigel until further assay. For TIGAR-induction, 4-OHT (Sigma, Cat#T176) with a concentration of 10 nM was administrated in vitro. After 24 h, the medium was replaced by normal organoid culture medium, with organoids still in Matrigel.

Chen F., Zhang Y., Hu S., Shi X., Wang Z., Deng Z., Lin L., Zhang J., Pan Y., Bai Y., Liu F., Zhang H, & Shao C. (2020). TIGAR/AP-1 axis accelerates the division of Lgr5− reserve intestinal stem cells to reestablish intestinal architecture after lethal radiation. Cell Death & Disease, 11(7), 501.

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Intestinal Organoid Irradiation Assay

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Small intestinal crypts were isolated and subsequently used for Western blot analysis or further culture. Isolated crypts were suspended with Matrigel (Corning, no. 356231) and incubated with advanced DMEM/F12 medium (Thermo Fisher Scientific, no. 12634-010) that was supplemented with EGF (Peprotech, no. 315-09-100), Noggin (Peprotech, no. 250-38-5), R-spondin (Peprotech, no. 315-32-5), N2 (Thermo Fisher Scientific, no. 17502048), B27 (Thermo Fisher Scientific, no. 17504044), and Y-27632 dihydrochloride monohydrate (Sigma, no. Y0503). After being cultured for 2 d in vitro, organoids were exposed to CONV or FLASH irradiation at different doses.

Shi X., Yang Y., Zhang W., Wang J., Xiao D., Ren H., Wang T., Gao F., Liu Z., Zhou K., Li P., Zhou Z., Zhang P., Shen X., Liu Y., Zhao J., Wang Z., Liu F., Shao C., Wu D, & Zhang H. (2022). FLASH X-ray spares intestinal crypts from pyroptosis initiated by cGAS-STING activation upon radioimmunotherapy. Proceedings of the National Academy of Sciences of the United States of America, 119(43), e2208506119.

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Enteroid Culture and Quantification

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For enteroid culture, approximately 40 freshly isolated jejunal crypts were suspended in 10 μL of growth factor reduced Matrigel (cat. #356231; Corning, Corning, NY) per well of a 24- well plate by using methods similar to those previously published by Sato et al.³ (link) After 30 minutes at 37°C to solidify the Matrigel, 200 μL of Wnt3a-free culture medium [DMEM/F12 Advanced media (cat. #12634-010; Thermo Fisher Scientific), 1× penicillin/streptomycin (cat. #15140-122; Thermo Fisher Scientific), 1× HEPES (cat. #15630-106; Thermo Fisher Scientific), 1× Glutamax (cat. # 35050-079; Thermo Fisher Scientific), 1× N2 (cat. # 17502-048; Thermo Fisher Scientific), 1× B27 (cat. #17504-044; Thermo Fisher Scientific), 50 ng/mL EGF (cat. #2028-EG; R&D Systems, Minneapolis, MN), 100 ng/mL Noggin (cat. #250-38; Peprotech, Rocky Hill, NJ), and 500 ng/mL R-spondin (cat. #4645-RS/CF; R&D Systems)] was added to each well. Media were changed every other day. For quantification of budding, enteroids were evaluated on days 1, 3, and 6 after plating for number of buds. Enteroids with no buds were defined as spheres. The remaining enteroids were grouped into categories of 1, 2, 3, or 4+ buds. Data are presented as percentage.

Dekaney C.M., King S., Sheahan B, & Cortes J.E. (2019). Mist1 Expression Is Required for Paneth Cell Maturation. Cellular and Molecular Gastroenterology and Hepatology, 8(4), 549-560.

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Isolation and Culture of Mouse Intestinal Organoids

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Isolation and culture of mouse small intestinal organoids, collected from the small intestinal mucosa, were carried out as described previously [21 ]. Briefly, isolated crypts were mixed with Matrigel (BD Biosciences, San Jose, CA) and cultured in basic culture medium (ENR) containing advanced DMEM/F12 medium with 50 ng/ml EGF (PeproTech), 100 ng/ml noggin (PeproTech), and 100 ng/ml R-spondin (PeproTech). The organoids were treated with TNFα or H₂O₂ for 48 h and total protein and RNA were extracted. All animal procedures were conducted in accordance with National Institutes of Health guidelines and were approved by the University of Kentucky Institutional Animal Care and Use Committee.

Kim J.T., Napier D.L., Kim J., Li C., Lee E.Y., Weiss H.L., Wang Q, & Evers B.M. (2021). Ketogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells. Free radical biology & medicine, 172, 90-100.

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Murine Intestinal Crypt Organoid Culture

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Murine intestinal crypt cultures were established from wild-type mice of a mixed background
(50% C57Bl6J, 50% S129) as previously described (Sato et al,
2009 (link)). Briefly, intestines were isolated and flushed with
PBS. Villi were removed by scraping and remaining tissue cut and washed with PBS. Crypt extraction
was carried out in PBS + 2 mM EDTA for 30 min at
4 °C. Isolated crypts were washed with PBS and plated in growth factor reduced
Matrigel (BD Biosciences). Crypts were grown in Advanced DMEM (Invitrogen) supplemented with N2
(Invitrogen), B27 (Invitrogen), EGF (Peprotech), Noggin (Peprotech), and R-spondin (Peprotech).
Media were replaced every 2 days and crypts passaged weekly. For drug treatments, crypts were
grown until they formed budding “organoids” and then treated with 10 μM
BI8622 or control treated with 0.1% DMSO for 24 h. Organoid survival was scored at
this point based on their microscopic appearance. Cell viability was also determined using a
CellTiter-Blue Cell Viability Assay (Promega) according to the manufacturer's
instructions.

Peter S., Bultinck J., Myant K., Jaenicke L.A., Walz S., Müller J., Gmachl M., Treu M., Boehmelt G., Ade C.P., Schmitz W., Wiegering A., Otto C., Popov N., Sansom O., Kraut N, & Eilers M. (2014). Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase. EMBO Molecular Medicine, 6(12), 1525-1541.

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Protocol for Intestinal Organoid Generation

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For generating intestinal organoids, crypts from C57BL6J mice were used. Mice were anesthetized using CO₂ and crypts were isolated from the small intestine by incubation of tissue for 25 min at 4 °C in crypt isolation buffer (CIB, PBSO containing 0.5 M EDTA). Isolated crypts were counted and a total of 500 crypts were plated with 25 µL Matrigel (Corning B.v., Amsterdam, Netherlands) in a 48-well plate. After polymerization of Matrigel, 300 µL of crypt culture medium (CCM) consisting of advanced DMEM/F12 (ThermoFisher Scientific, Karlsruhe, Germany) was supplemented with 100 ng/µL Noggin (PeproTech, East Windsor, NJ, USA), 1 µg/mL R-Spondin (PeproTech, East Windsor, NJ, USA), B-27™ supplement 1× (Invitrogen, Carlsbad, CA, USA), 1 mM N-Acetylcystein (Sigma-Aldrich, Darmstadt, Germany), 0.1 mg/mL Primocin (Invitrogen, Carlsbad, CA, USA) and 50 ng/mL recombinant mouse epidermal growth factor (rm EGF) (Immunotools, Friesoythe, Germany). The resulting organoids were cultured and splitted for a minimum of seven days according to Sato et al. 2009 [33 (link)].

Filipe Rosa L., Petersen P.P., Görtz L.F., Stolzer I., Kaden-Volynets V., Günther C, & Bischoff S.C. (2023). Vitamin A- and D-Deficient Diets Disrupt Intestinal Antimicrobial Peptide Defense Involving Wnt and STAT5 Signaling Pathways in Mice. Nutrients, 15(2), 376.

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