For construction of organoids, 200–500 crypts per well were suspended in Matrigel (Corning) as described.58 (link) Complete ENR medium (all components from Thermo Fisher Scientific unless noted) were comprised of advanced DMEM/F12 (Gibco), antibiotic-antimycotic (×100), 1 mM N-acetyl cysteine (Sigma-Aldrich), B27 supplement, N2 supplement, E GF, N oggin (R&D Systems), R -spondin-1-conditioned medium, and Y-27632 (Sigma). Y-27632 was added to the ENR medium for the first 48–72 h of culture only and then removed during the medium change. The ENR medium was replaced every 2 to 3 days. Colon organoids were cultured in the ENR medium supplemented with Wnt3 conditioned media (WENR). Human colon organoids were cultured in WENR medium supplemented with gastrin, nicotinamide, A83-01, and SB202190 (all from Sigma) as described.59 (link) Isolated ISCs and Paneth cells were co-cultured in ENR medium supplemented with Jagged-1 (1 µM; Anaspec). Wnt-C59 (50 µM; Abcam) was used as a porcupine (PORCN) inhibitor. The surface areas of SI and colon organoids were measured microscopically by taking several random non-overlapping photos of organoids in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss). Organoid perimeters for area measurements were defined manually using automated ImageJ software.
Jagged 1
Manufactured by AnaSpec
Sourced in United States, Japan
Jagged-1 is a recombinant protein produced by AnaSpec. It is a key signaling molecule involved in the Notch signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
R spondin 1, by R&D Systems (5 mentions)
Y 27632, by Merck Group (4 mentions)
Matrigel, by Corning (4 mentions)
Noggin, by Thermo Fisher Scientific (4 mentions)
Matrigel, by BD (4 mentions)
Y 27632, by Fujifilm (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Wnt3a, by R&D Systems (2 mentions)
Rock inhibitor, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by BD (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
Sb202190, by Merck Group (1 mentions)
N2 supplement, by R&D Systems (1 mentions)
B 27 supplement, by R&D Systems (1 mentions)
Zen image program, by Zeiss (1 mentions)
Noggin, by R&D Systems (1 mentions)
A83 01, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
16 protocols using jagged 1
1
Organoid Culture from Intestinal Crypts
2
Intestinal Stem Cell Niche Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal number of ISCs Lin−CD24med/loEGFPhi and Paneth cells Lin−EGFPnegCD24hiSSChi cells were mixed and cultured in 60% Matrigel overlaid with ENR media supplemented with extra R-spondin1 (500 ng/ml), Wnt3A (100 ng/ml; R&D Systems), and 1 μM Jagged-1 (AnaSpec). Media contained 10 μM Y-27632 for the first 2 days. Alternatively, for colony formation assay, single ISCs alone were cultured in ENR media supplemented with 10 μM Chir99021 and 10 μM Y-27632. Medium was changed every 2 days, cocultures were quantified on day 6, and colony cultures were quantified on day 5.
3
Organoid Culture and Labeling Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were cultured in standard cultured medium for 7 days and subsequently enzymatically passaged into single cells using TrypLE express, as described above for flow cytometry. The single cells were subsequently stained with green CMFDA (5, 15, or 25 μM), after which they were washed and homogenized in diluted Matrigel®, supplemented with Jagged-1 (1 μM) (AnaSpec, Fremont, CA, USA) [23 (link)] and seeded in triplicates. Cells were cultured in standard medium (without supplementation of ROCK inhibitor) for a duration of 10 days. At day 10, the number of organoids was determined manually using an inverted digital light microscope. Six experiments were performed in total and included unstained DMSO (dimethylsulfoxid)-control samples as well as cells that briefly were treated with Triton™ X-100 (5%) (Sigma-Aldrich).
4
Uterine Horn Organoid Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bilateral uterine horns were isolated from C57BL/6 J mice at approximately 10 weeks of age. The tissues were minced into 2–3 mm pieces, washed several times with cold PBS, and dissociated with 2 U/mL dispase II and 1 mg/mL collagenase P (Roche Diagnostics K.K., Tokyo, Japan) for 30 min at 37 °C. Dissociated cells were subjected to the Matrigel bilayer organoid culture (MBOC) protocol [22 (link)]. Briefly, cells were resuspended in the medium supplemented with L-glutamine solution (Wako, Osaka, Japan), penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO), amphotericin B suspension (Wako), 50 ng/mL EGF (Peprotech, Rocky Hill, NJ), 250 ng/mL R-spondin1 (R&D, Minneapolis, MN), 100 ng/mL Noggin (Peprotech), 10 μM Y27632 (Wako, Osaka, Japan), 1 μM Jagged-1 (AnaSpec, Fremont, CA), and 2.5 μM CHIR-99021 (Cayman Chemical, Ann Arbor, MI), and plated on 65 μL of solidified Matrigel (BD Biosciences, Franklin Lakes, NJ) per well in a 12-well plate. After overnight incubation at 37 °C, floating or dead cells were removed, leaving only viable cells attached onto Matrigel, which were covered with 70 μL of Matrigel and the medium. Passage was conducted every 5–10 days at 1:2 to 1:3 dilution. In each passage, organoids were dissociated using Accutase (Innovative Cell Technologies, San Diego, CA) for 5 min at 37 °C and vigorous pipetting, followed by seeding on Matrigel.
5
Intestinal Organoid Culture Technique
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were cultured from crypt enriched ileal fractions from
6–10 wk old wildtype and
Mmp7−/− mice as previously
described1 (link). Briefly,
the distal 10 cm of the small intestine was removed and flushed with
0.04% sodium hypochlorite in PBS. After removal of mucus and villi, the
epithelium was dissociated for 90 min at RT in a solution of 3 mM EDTA and 0.5
mM DTT in Hank’s Buffered Salt solution (HBSS). Crypt enriched fractions
were identified following vigorous shaking into sequential changes of fresh, ice
cold, sterile Ca2+/Mg2+-free HBSS. Cells
were then concentrated by centrifugation at 300 × g for 5 min at 4
°C, and the pellet was resuspended in 300 μl of HBSS containing
0.5 mM Rock inhibitor (Fisher) and 10 μM Jagged-1 (Anaspec). After a
second round of centrifugation, the cell pellet was resuspended in growth factor
reduced Matrigel (BD Biosciences). 50 μl aliquots were plated in the
center of 24 well plates and overlayed with 500 μl of Complete Crypt
Culture Medium (CCCM)10 (link). Once
established, culture media was supplemented with 200 μl CCCM every
2–3 days. Organoids were subcultured every 6–7 days10 (link).
6–10 wk old wildtype and
Mmp7−/− mice as previously
described1 (link). Briefly,
the distal 10 cm of the small intestine was removed and flushed with
0.04% sodium hypochlorite in PBS. After removal of mucus and villi, the
epithelium was dissociated for 90 min at RT in a solution of 3 mM EDTA and 0.5
mM DTT in Hank’s Buffered Salt solution (HBSS). Crypt enriched fractions
were identified following vigorous shaking into sequential changes of fresh, ice
cold, sterile Ca2+/Mg2+-free HBSS. Cells
were then concentrated by centrifugation at 300 × g for 5 min at 4
°C, and the pellet was resuspended in 300 μl of HBSS containing
0.5 mM Rock inhibitor (Fisher) and 10 μM Jagged-1 (Anaspec). After a
second round of centrifugation, the cell pellet was resuspended in growth factor
reduced Matrigel (BD Biosciences). 50 μl aliquots were plated in the
center of 24 well plates and overlayed with 500 μl of Complete Crypt
Culture Medium (CCCM)10 (link). Once
established, culture media was supplemented with 200 μl CCCM every
2–3 days. Organoids were subcultured every 6–7 days10 (link).
6
Gastric Organoid Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoid cultures of gastric epithelial cells and gastric tumors were performed as previously described23 (link). For primary gastric epithelial cell cultures, glandular stomachs of mice were treated with 0.1% collagenase for 30 min followed by trypsin digestion, and cells were cultured in Matrigel (BD Biosciences, Franklin Lakes, NJ, USA & CORNING) with the primary culture medium containing 50ng/mL EGF (BD Biosciences) supplemented with 500 ng/mL R-spondin1 (R&D, Minneapolis, MN, USA), 2.5 ng/μl of Jagged1 (AnaSpec, Fremont, CA, USA), and 100 ng/mL of rMuNoggin (PeproTech, Rocky Hill, NJ, USA). The first primary cultured cells were cultured with CHIR99021 (2.5 μM; Stemgent, Cambridge, MA) at day 031 . The mean number of cystic structures >300 μm in diameter in Matrigel were counted at day 5~9.
7
Organoid Culture of Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bile and biopsy samples were collected endoscopically. The tumor was surgically resected. These specimens were subjected to MBOC as previously described.34 Briefly, enzymatically dissociated cells were embedded in a bilayer of Matrigel (BD Biosciences, Franklin Lakes, NJ) by two‐step procedures42 and maintained in advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with DGF including 50 ng/ml human epidermal growth factor (EGF) (Peprotech), 250 ng/mL R‐spondin1 (R&D), 100 ng/ml Noggin (Peprotech), 10 μM Y27632 (Wako, Osaka, Japan), and 1 μM Jagged‐1 (AnaSpec).
8
Comprehensive Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HGF (R&D), Dkk1 (R&D), Wif1 (R&D), Draxin (R&D), sFRP1 (R&D), sFRP5 (R&D), BMP4 (R&D), Gremlin (R&D), Jagged 1 (Anaspec), BMP3 (R&D), BMP7 (R&D), Dll1 (R&D), Dll4 (R&D), DAPT (Sigma), DAPT-GSI (Sigma), TGF-α (R&D), Lrig1 (R&D), Shh (R&D), Ihh (R&D), Itraconazole (Sigma), Gant-61 (Tocris Bioscience), Calcitriol/Vit D (Sigma), Rapamycin (LC Laboratories), ICG-001 (Tocris), and Carnosic Acid (Sigma; also see Data S1)
Xenograft and in vivo Itraconazole dosing experiments: Beacon Pharmaceuticals (10 mg/ml).
Antibodies for IHC and FACS: AGR2 (Atlas, HPA007912), GDF15 (Atlas, HPA011191), β-catenin (BD Biosciences, 610154), and PTK7 (clone 188B; Miltenyi).
Xenograft and in vivo Itraconazole dosing experiments: Beacon Pharmaceuticals (10 mg/ml).
Antibodies for IHC and FACS: AGR2 (Atlas, HPA007912), GDF15 (Atlas, HPA011191), β-catenin (BD Biosciences, 610154), and PTK7 (clone 188B; Miltenyi).
9
Isolation and Culture of Primary Cells from Tumor, Normal Adjacent, and Healthy Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All tissues were excised after surgery, stored and transported in wash buffer: F12 (Gibco), 5% FBS (Hyclone), 1% penicillin/streptomycin (Gibco), 0.1% Amphotericin B (Gibco), 0.25% Gentamicin (Gibco), 1% HEPES (Gibco), and 5 μM Rock inhibitor (Calbiochem) at 4 °C. All tissues were cut into small pieces and incubated in 1 mg/ml collagenase type XI buffer (Gibco) at 37 °C, but incubation time was different. 15 min for tumor tissue, 60 min for NAT and HLT. The digested cell solution was filtered through a 70 μm cell strainer (Falcon), and washed four times with a wash buffer. Isolated cells were resuspended in stem cell medium (SCM): Advanced DMEM/F12 (Hyclone) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-Glutamine (Hyclone), 0.1% Amphotericin B, 0.5% Gentamicin, 0.18 mM Adenine (Sigma), 5 μg/mL Insulin (Sigma), 2 nM T3 (Sigma), 200 ng/mL Hydrocortisone (Sigma), 125 ng/mL R-Spondin 1(R&D), 1 μM Jagged-1(AnaSpec Inc), 100 ng/mL Noggin (Peprotech), 2.5 μM Rock inhibitor (Calbiochem), 2 μM SB431542 (Cayman chemical), 10 mM Nicotinamide (Sigma), and 10 ng/mL EGF (Upstate Biotechnology). After resuspension, the cells were seeded into irradiated 3T3-J2 feeder cells that were paved one day in advance, and cultured at 37 °C in 7.5% CO2. The culture medium was replaced every two days.
10
Organoid Culture with Growth Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were cultured as described previously with slight modifications37 (link). Sorted EpCAM+ cells were mixed with Matrigel (Corning, Corning, NY) containing 750 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ), 1.5 μg/mL Noggin (Miltenyi Biotec, Germany), and 15 μmol/L Jagged-1 (Anaspec, Fremont, CA). The cells were seeded on 96-well plates at 3,000 cells/10 μL of Matrigel per well. After the Matrigel polymerized, 100 μL of culture medium consisting of Advanced Dulbecco’s Modified Eagle Medium/F12 supplemented with 100 U/mL penicillin/streptomycin, GlutaMAX, 10 mmol/L HEPES, 1 × N2, 1 × B27, 1 mmol/L N-acetylcysteine, 2.5 μmol/L Thiazovivin (Cayman, Ann Arbor, MI), and 2.5 μmol/L CHIR99021 (Axon Medchem, The Netherlands) was added to each well. On day 2 of culture, 50 μL of culture medium containing 10% (v/v) R-spondin 1 culture supernatant and growth factors (1 μM Jagged-1, 50 ng/mL epidermal growth factor, and 100 ng/mL Noggin at final concentration) was added to each well. On day 6, the number of organoids was counted and photographed in each well. The R-spondin 1 culture supernatant was harvested from the 293T-HA-RspoI-Fc cell line, which was provided by Calvin Kuo of Stanford University.
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!