Control (n = 3) and D164A (n = 3) mice have sacrificed 0, 2 or 4d post injection, crypts were isolated as described50 (link). Crypt fractions 3 and 4 were pooled and dissociated to single cells by incubating them with 5 mL pre-warmed TripLE Express Enzyme (ThermoFischer) in a 37 °C water bath for 5 min with frequent agitation. TripLE was inactivated with 50% FBS in Advanced DMEM/F12 (ThermoFischer). Single cells were pelleted at 180 × g for 3 min and resuspended in 2 mL cold Intesticult (Stemcell Technologies Inc). Clumps were dissolved by pipetting up and down with a 1 mL pipet before filtering twice through a 40 μm filter. Cell viability and number were quantified in automatically with a Countess (Thermo Scientific). DropSeq workflow was performed as described62 on a Nadia Instrument (Dolomite Bio). Sequencing was performed on the Illumina NovaSeq6000 with an SP Reagent Kit.
Intesticult
Manufactured by STEMCELL
Sourced in Canada, United States
Intesticult is a comprehensive kit that enables in vitro culture of intestinal organoids. The kit includes essential reagents and media for the isolation, expansion, and maintenance of intestinal stem cells and their differentiation into mature intestinal cell types.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (6 mentions)
Primocin, by InvivoGen (3 mentions)
Celltiter glo luminescence assay, by Promega (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
A83 01, by Merck Group (2 mentions)
Gastrin 1, by Merck Group (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sp reagent kit, by Illumina (1 mentions)
Triple express enzyme, by Thermo Fisher Scientific (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Fgf10, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
16 protocols using intesticult
1
Dropseq Analysis of Mouse Intestinal Crypts
2
Murine Intestinal Organoid Culture
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Crypt isolation and organoid cultures from murine small bowel were performed based on the methods of Mahe and colleagues [6 (link)] and using the Intesticult™ system from StemCell Technologies (Vancouver, Canada) according to manufacturer’s instructions. Small intestines were isolated from mice and crypts isolated as described. 300 crypts were seeded in 50 µL mixture of Matrigel and Intesticult™ medium in 24-well plates. For MTT assay 150 crypts were seeded in 25 µL mixture in 48-well plates. Diphenyleneiodonium (DPI) and 4-OHT were added to the medium after seeding in indicated concentrations and removed after 48 h. On day 4 after isolation organoids were analyzed. To isolate organoids from the matrigel and prepare them for further analysis, TrypLE© Express (Life Technologies, Carlsbad, CA, USA) was added to the matrigel domes. Matrigel was digested for 10 min at 37 °C. The reaction was stopped by dilution with Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Waltham, MA, USA) and the organoids were processed for further analysis.
3
Organoid growth media optimization
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In addition to the basic growth medium, termed L-WRN medium, described above, we also tested a commercially available growth medium, IntestiCult™ (StemCell), a complex medium termed “colonoid medium” described by Tsai et al.86 (link), and analyzed medium supplementation with a number of different growth factors commonly used in organoid culture protocols (Supplementary Table 1 ). We prepared a medium with all available growth factors (L-WRN Plus) and then eliminated one reagent at a time from L-WRN Plus to determine the influence of the reagent on organoid growth. For this assay, the organoids were digested with TrypLE for 3 min and plated in a 96-well plate with the different media. Cell viability and proliferation were measured using the CellTiter-Glo luminescence assay (Promega).
4
Gallbladder Cancer Organoid Culture
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GBC-PDOs cultures were established from tumor tissue samples of patients diagnosed with moderated differentiated and pT3 N0/N1 Mx stage (Eight edition of American Joint Committee on Cancer (AJCC)) advanced tubular GBC. Gallbladder tumor organoids were generated as previously described [54 (link)]. For organoids cultures, dissociated cells were washed in 1× DPBS and embedded in growth factor reduced Matrigel (#356231, Corning, NY, USA) and cultured in complete media (Intesticult (#6005, Stemcell Technologies, Vancouver, BC, Canada), 0.5 μmol/L A83-01 (#SML0788; Sigma-Aldrich), 100 ng/mL, FGF10 (#100-26, PeproTech, Inc., Rocky Hill, NJ, USA), 100 ng/mL HGF (#100-39; Peprotech), 10 nmol/L Gastrin I (#G9145; Sigma), 10 mmol/L N-acetyl-l -cysteine (#A9165; Sigma-Aldrich) 10 mmol/L nicotinamide (#N0636; Sigma-Aldrich), 1× B27 supplement (#17504-044; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1× N2 supplement (#175020-048; Gibco), 1 mg/mL Primocin (#ant-pm-1; InvivoGen, San Diego, CA, USA) and 10.5 μmol/L Y-27632 (#Y0503, Sigma-Aldrich). Organoids were passaged via mechanical dissociation and passage was performed weekly with a 1:3 ratio.
5
Isolation and Culture of Murine Colonoids
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Mice were killed and colon was collected, cut open longitudinally in a Petri dish with cold PBS without Ca2+/Mg2+ (Sigma-Aldrich), vigorously washed using forceps, and cut in 2- to 5-mm pieces. Next, the pieces were collected in a 50-mL Falcon tube filled with 30 mL Dulbecco′s Phosphate Buffered Saline (DPBS) plus 120 μL ethylenediaminetetraacetic acid 0.5 mol/L (Sigma-Aldrich) and incubated for 45 minutes at 4ºC. Subsequently, colon pieces were collected in 4 fractions of 25 mL DPBS. The 4 fractions were inspected under a microscope (10× objective) to identify the fractions enriched in crypts and with low level of debris. The chosen fraction was spun down (450 × g for 10 minutes at 4ºC). The supernatant was removed and the pellet was resuspended in 150 μL Intesticult (STEMCELL Technologies) and 150 μL Matrigel basement membrane matrix (Corning, Inc, Corning, NY). Aliquots of 40 μL of this solution then were plated in a prewarmed 48-well plate (VWR, Radnor, PA) into the center of the well and incubated for 10 minutes at 37ºC, allowing the Matrigel to polymerize. Finally, 350 μL Intesticult was added in each well of the plate. Colonoids were cultured for 10 days and passaged once after 6 days. Total RNA was extracted on day 10 and gene expression was measured by NanoString analysis.
6
Isolation and Culture of Human Intestinal Organoids
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Fresh tissue from human jejunum
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
7
Culturing Mouse Intestinal Organoids
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Mouse intestinal organoids were cultured in a mixture of Matrigel (Corning, New York, NY, USA) and organoid growth medium (Intesticult; STEMCELL Technologies). Briefly, organoid pellets were suspended in a mixture of Matrigel and Intesticult at a ratio of 1:1. The organoids were then seeded in 6-well plates, with 4–5 Matrigel domes per well. The plates were incubated at 37 °C for 20 min to allow Matrigel solidification, and then 2 mL Intesticult was added to each well. The culture was maintained for 7–14 days before the next passage.
8
Porcine Intestinal Organoid Culture
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As with the bovine tissue, sections of proximal jejunum (approximately 10 cm in length) from adult pigs were obtained from an FSA approved slaughterhouse within 10 miles of the laboratory. Tissue was acquired following the procedures and restrictions described in the Derogations from Animal By-Product controls under Regulation (EC) 1069/2009 and Commission Regulation (EU) 142/2011 (DEFRA 2011 ). Tissue was prepared for crypt isolation as described previously for bovine tissue (Fig. 1 ). We calculated that 0.5–1 ml of crypt suspension was required for each well of organoids to be cultured. The required volume of crypt suspension was centrifuged at 125g for 7 min at 4 °C, the supernatant aspirated then the crypt pellet resuspended in a pre-prepared mix of 70% Matrigel (Corning), 30% IntestiCult (Stem Cell Technologies) with 100 μg/ml Primocin (InvivoGen) and 30 μl applied on coverslips in a pre-warmed 48-well plate. Following 30-min Matrigel polymerisation time at 37 °C, 200 μl of IntestiCult at RT was added to each well. Cultures were incubated at 37 °C, 5% CO2. Medium was changed every 3–4 days until the organoids required passage (~ 7 days).
9
Organoid Culture of Intestinal Crypts
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Surgical specimens from the healthy ileum of CD patients of patients with right colon cancer were used. Briefly, the mucosa was stripped from the submucosa cut into small pieces and incubated sequentially in an antibiotic solution, then in DL-Dithiothreitol (DTT) solution and in an Ethylene Diamine Tetra-acetic Acid (EDTA) solution at 4°C. Samples were then immerged in cold PBS and shaken vigorously to extract the intestinal crypts.
Crypts were cultivated in Matrigel (Corning) drops at the concentration of approximately 100 crypts per drop. Intesticult (STEM CELL Technologies), supplemented with Y-27632 (STEM CELL Technologies), was then added. Medium was first changed at 24 hours with Y-27632 free-Intesticult and then every two days.
After 5 days of crypt culture, organoids were formed and ready to be co-cultivated. The day before the planned co-culture selected wells were incubated overnight with a LPS solution at the concentration of 1 μg/ml.
Crypts were cultivated in Matrigel (Corning) drops at the concentration of approximately 100 crypts per drop. Intesticult (STEM CELL Technologies), supplemented with Y-27632 (STEM CELL Technologies), was then added. Medium was first changed at 24 hours with Y-27632 free-Intesticult and then every two days.
After 5 days of crypt culture, organoids were formed and ready to be co-cultivated. The day before the planned co-culture selected wells were incubated overnight with a LPS solution at the concentration of 1 μg/ml.
10
Irradiation-Induced Intestinal Enteroid Analysis
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