Crypts were purified from mice as previously described (Sato et al, 2009 (link)). Crypts were seeded in growth factor-reduced Matrigel (BD Biosciences) with the addition of EGF and Noggin (both Peprotech), without R-spo1 or with 50 ng/ml R-spo1 (R&D Systems). About 300 crypts were plated per 20 μl matrigel/well, and procedures were carried out within 3 h after sacrificing the mouse. Representative photographs were taken after 7 days.
Rspo1
Manufactured by R&D Systems
Sourced in United States
Rspo1 is a recombinant protein product manufactured by R&D Systems. It functions as an agonist for the Wnt signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (8 mentions)
Noggin, by Thermo Fisher Scientific (6 mentions)
Rock inhibitor y 27632, by Merck Group (6 mentions)
Noggin, by R&D Systems (4 mentions)
Matrigel, by BD (3 mentions)
Fgf10, by Thermo Fisher Scientific (3 mentions)
Recombinant human wnt3a, by R&D Systems (3 mentions)
Neutralization antibody for rspo1, by Abcam (3 mentions)
Epidermal growth factor (egf), by BD (3 mentions)
Matrigel, by Corning (2 mentions)
Gastrin, by Merck Group (2 mentions)
Y 27632, by Merck Group (2 mentions)
Ade cre, by Vector Biolabs (2 mentions)
Tryple select, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Hbss buffer, by Thermo Fisher Scientific (2 mentions)
Ade cre, by Corning (2 mentions)
Dmem f12, by STEMCELL (2 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
18 protocols using rspo1
1
Culturing Mouse Intestinal Crypts
2
Enteroid Isolation and Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enteroids were established from freshly isolated proximal small intestinal crypts according to standard protocols.54 (link) Crypts were seeded in 50 μL growth factor reduced Matrigel (Corning Life Sciences, Corning, NY) drops at 1000 crypts/mL into pre-warmed 24-well tissue culture plates and then overlaid with enteroid culture media (Advanced DMEM/F12 + 10 mmol/L HEPES, 4 mmol/L L-glutamine, 1× N2 supplement and B27 supplements [and 1× penicillin/streptomycin], all from Gibco Laboratories, Gaithersburg, MD), and the following growth factors: Jagged-1 peptide (1 μmol/L; Anaspec, Fremont, CA), Egf (50 ng/mL; R&D Systems, Minneapolis, MN), Noggin (100 ng/mL; Peprotech, Rocky Hill, NJ), and Rspo1 (1 μg/mL; R&D Systems). Enteroids were imaged by using either a Leica DMIRB or Nikon Eclipse TE200 inverted microscope. Live imaging was performed by using an Incucyte ZOOM microscope system (Essen BioScience Inc, Ann Arbor, MI). Enteroid growth and budding were quantified from images by using ImageJ (National Institutes of Health, Bethesda, MD).
3
Colorectal Adenoma Organoid Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human adenoma biopsy samples were obtained from patients (40–70 years old) for research purpose under the approval of the Ethical Committee of Medical Research, Huashan Hospital of Fudan University (2018-182). Informed consent had been obtained from all subjects involved.
Human colorectal adenoma samples were minced into 3 mm3, then washed with PBS three times. The tissue fragments were digested by collagenase solution (collagenase 0.125 mg/mL, dispase 0.5 mg/mL, FBS 1% in DMEM medium) for 50 min at 37 °C. Isolated epithelium was mixed with Matrigel (Corning) and cultured as previously described [19 (link)]. Briefly, CA organoid culture medium was the basal medium supplemented with 500 ng/mL RSPO1 (R&D system, Minneapolis, MN, USA), 100 ng/mL Noggin (R&D), 50 ng/mL EGF (Invitrogen, Carlsbad, USA), 10 nM gastrin (Sigma-Aldrich, St Louis, MO, USA), 500 nM A83-01, and 10 μM SB202190. In order to determine the optimum culture medium with selectivity for CA organoids, we compared CA medium with or without 10 ng/mL Wnt3a. Culture medium was changed every other day. Within 1 week, organoids were passaged at a 1:3 ratio with TrypLE digestion.
Human colorectal adenoma samples were minced into 3 mm3, then washed with PBS three times. The tissue fragments were digested by collagenase solution (collagenase 0.125 mg/mL, dispase 0.5 mg/mL, FBS 1% in DMEM medium) for 50 min at 37 °C. Isolated epithelium was mixed with Matrigel (Corning) and cultured as previously described [19 (link)]. Briefly, CA organoid culture medium was the basal medium supplemented with 500 ng/mL RSPO1 (R&D system, Minneapolis, MN, USA), 100 ng/mL Noggin (R&D), 50 ng/mL EGF (Invitrogen, Carlsbad, USA), 10 nM gastrin (Sigma-Aldrich, St Louis, MO, USA), 500 nM A83-01, and 10 μM SB202190. In order to determine the optimum culture medium with selectivity for CA organoids, we compared CA medium with or without 10 ng/mL Wnt3a. Culture medium was changed every other day. Within 1 week, organoids were passaged at a 1:3 ratio with TrypLE digestion.
4
Establishment of Lgr5+ Liver Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice or human Lgr5+ liver cells were isolated. Single Lgr5-GFP+ liver cells were mixed with Matrigel (BD Bioscience) and cultured, as described in ref. 8 (link). The medium composition was as follows: AdDMEM/F12 (Invitrogen) supplemented with 1% B27 and 1% N2 (Invitrogen), N-acetylcysteine (1.25 mM, Sigma-Aldrich), gastrin (10 nM, Sigma), EGF (50 ng/ml, Peprotech), Rspo1 (50 ng/ml, R&D), FGF10 (100 ng/ml, Peprotech), nicotinamide (10 mM, Sigma-Aldrich), and HGF (50 ng/ml, Peprotech). The medium was changed every other day. For human Lgr5+ liver cells culture, 5 µM A83.01 (Tocris), and 10 µM FSK (Tocris) were added into the medium22 (link). For the establishment of the culture, the medium was supplemented with 25 ng/ml Noggin (R&D), 25 ng/ml Wnt (R&D), and 10 µM Y27632 (Sigma-Aldrich) for the first 3 days after isolation. Next, the medium was changed into a medium without Noggin, Wnt, and Y27632. After 10–14 days, organoids were removed from Matrigel, mechanically dissociated into small fragments, and transferred to a fresh matrix. A passage was performed in a 1:4–1:8 split ratio once every 7–10 days for at least 6 months.
5
Modulating eMSC Differentiation with WNT3A and RSPO1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutralization antibody for RSPO1 (1 μg/ml, Abcam) was added to the epithelial CCM from the menstrual phase (1/3-unit: growth medium). Isotype antibody rabbit IgG was used as negative control. Recombinant human WNT3A (12.5, 25, 50 ng/ml, R&D Systems) and RSPO1 (50 ng/ml, R&D Systems) was supplemented to the growth medium of eMSCs seeded at clonal density for 15 days.
6
Ade-Cre Mediated Genetic Manipulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ade-Cre were purchased from Vector Biolabs. MECs or FACS-sorted basal MECs were plated at 10 million/mL/well in 24-well plates in DMEM/F12 (Stemcell Technologies, 36254) containing 2% FBS, 10 mM HEPES (Millipore Sigma, H3375), 10 ng/ml EGF (Invitrogen, PMG-8043), 250 ng/ml Rspo1 (R&D, 3474-RS), 100 ng/ml Noggin (Fisher Scientific, 50–399-006), and 10 μM Rock inhibitor Y27632 (Millipore Sigma, SCM075), and cultured overnight. Cells were then infected with Ade-Cre at a multiplicity of infection (MOI) of 10 for overnight in 24-well ultra-low attachment surface polystyrene plate (Corning, REF 3473). Cells were harvested the next day, and treated with 400 μL TrypLE Select (GIBCO, 12605–010) for 20 min at 37°C followed by neutralization with 2 mL HBSS buffer (GIBCO, 14025134). GFP+ cells were FACS-sorted for ex vivo culture or for transplantation.
7
Organoid Culture Conditions for Wnt Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The conventional medium was composed of DMEM supplemented with 1000 ng/ml of
Wnt3a, 200 ng/ml of RSPO1 (R & D, Minneapolis, MN, USA), 1% glutamax, 12 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1% N2, 2% B27, 10
ng/ml of EGF (Invitrogen), 100 ng/ml of FGF10 (Peprotech), 100 ng/ml of noggin,
1 mM of nicotinamide, 0.5 μM of TGF-β R kinase inhibitor IV (SB431542, Sigma),
and 9 μM of ROCK inhibitor (Y-27632, Sigma).
Wnt3a, 200 ng/ml of RSPO1 (R & D, Minneapolis, MN, USA), 1% glutamax, 12 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1% N2, 2% B27, 10
ng/ml of EGF (Invitrogen), 100 ng/ml of FGF10 (Peprotech), 100 ng/ml of noggin,
1 mM of nicotinamide, 0.5 μM of TGF-β R kinase inhibitor IV (SB431542, Sigma),
and 9 μM of ROCK inhibitor (Y-27632, Sigma).
8
Organoid Culture and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For organoid culture, 25,000 FTECs were cultured in 6 well dishes pre-coated with Matrigel (50 μl of 1% Matrigel (BD Matrigel Basement Membrane Matrix)) at 37 °C in a 5% CO2 atmosphere and overlaid with 500 μl organoid culture medium. The organoid culture medium consisted of DMEM supplemented with 50 ng/ml Wnt3a, 50 ng/ml RSPO1 (R & D, Minneapolis, MN, USA), 12 mM HEPES, 1% glutamax, 2% B27, 1% N2, 10 ng/ml EGF (Invitrogen), 100 ng/ml noggin, 100 ng/ml FGF10 (Peprotech), 1 mM nicotinamide, 9 μM ROCK inhibitor (Y-27632, Sigma), and 0.5 μM TGF-β R kinase inhibitor IV (SB431542, Sigma). After more than 21 days of culture, organoids were sent to immunohistochemistry for FOXJ1, detyrosinated TUBULIN (ciliated cell markers), PAX8 (a secretory cell marker), and vimentin (a mesoderm marker).
9
Wnt Signaling Modulation in Thyroid Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wnt signaling was assessed at baseline in Nthy-ori-3-1, TPC-1 and KTC-1 cell lines. Human cell lines were treated with recombinant human R-Spondin1 (RSPO1, R&D Systems, 4645-RS-025) at varying concentrations in a dose-dependent manner (0 ng/ml, 50 ng/ml, and 150 ng/ml) and harvested after 48 hours for mRNA and protein. Additionally, cell lines were treated with increasing doses of niclosamide (0 μM, 0.3 μM, and 1 μM), a Wnt/β-catenin antagonist, and harvested at 48 hours. All drug doses were consistent with published literature utilizing oncogenic cell lines [30 (link)].
10
Isolation and Culture of Intestinal Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The entire small intestine from 8- to 12-week-old male and female mice was collected, and then the duodenum was removed. The remaining tissue was flushed with cold PBS, cleaned of the mesentery, and opened lengthwise. EDTA-based (15575020; Invitrogen) dissociation was performed as previously described.69 (link) After EDTA dissociation, crypts were resuspended in Advanced Dulbecco’s modified Eagle medium/F12 (12634-010; Gibco) supplemented with GlutaMAX (35050061; Thermo Fisher Scientific), HEPES (15-630-080; Thermo Fisher Scientific), antibiotic–antimycotic (15240062; Fisher Scientific), 10% fetal bovine serum (900-108; Gemini Bio-Products), B27 (17504044; Thermo Fisher Scientific), N-2 supplement (17502048; Fisher Scientific), N-acetyl-L-cysteine (A9165; Sigma-Aldrich), EGF (PMG8043; Thermo Fisher Scientific), Noggin (250-38; PeproTech), Rspo1 (3474-RS-050; R&D Systems), and CHIR (SML1046; Sigma-Aldrich). Crypts were counted under a microscope, and then 200 crypts were seeded into 20 uL GFR Matrigel (356231; Corning) and plated in a prewarmed 24-well plate as domes. dmPGE2 and/or EP4i treatment began 24 hours after plating, and media was replaced every 2 days. Organoid size and number data were collected on day 6 after plating.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!