Colonoids used for sequencing are described in Supplemental Table 1 . Colonoids in Matrigel were treated overnight (18 h) with 50 μg/ml UBD. Next day, colonoids were removed from Matrigel by incubation with Organoid Harvesting Solution at 4°C for 45 min, washed in Advanced DMEM/F12 and pelleted by centrifugation at 300 xg for 10 min. Colonoids were resuspended in 500μl TrypLE enzyme solution (ThermoFisher), then transferred to a 24 well plate and spun at 600 rpm at 37°C for 45 min in a spinoculator (Mixer HC, USA scientific) to digest into single cells. Single cells were washed in 10 ml of Advanced DMEM/F12, pelleted again, resuspended in 1 ml of organoid expansion media and filtered with 40 μm Flowmi cell strainers (SP Bel-Art, Wayne, NJ). Cells were transported to the UNM Analytical and Translational Genomics Core for counting and processing. Average processing time from colonoids to library preparation was < 2.5 hours and the cells were highly viable (>90%).
Tryple enzyme solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
TrypLE enzyme solution is a liquid formulation of the proteolytic enzyme trypsin. Trypsin is commonly used in cell culture applications to gently dissociate adherent cells from a surface or container. The TrypLE solution provides a reliable and consistent enzymatic activity for cell detachment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (2 mentions)
Cell recovery solution, by Thermo Fisher Scientific (2 mentions)
William s e medium, by Thermo Fisher Scientific (2 mentions)
Mixer hc, by USA Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Evos cell imaging systems microscope, by Thermo Fisher Scientific (1 mentions)
Cell maintenance supplement pack, by Thermo Fisher Scientific (1 mentions)
Primary hepatocyte maintenance supplements, by Thermo Fisher Scientific (1 mentions)
6 protocols using tryple enzyme solution
1
Single-Cell Isolation from Colonoids
2
Colonoid Sequencing: Uranium Exposure
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Colonoids used for sequencing are described in Table S1. UBD was diluted in organoid expansion media, then colonoids in Matrigel were treated overnight (18 h) with UBD, with control colonoids receiving the same volume/volume of organoid expansion medium as the vehicle. Dosage was chosen based on prior studies26 (link),48 (link)–51 (link) that showed molecular changes to cells or animals exposed to uranium, but with minimal cytotoxic damage, since acute uranium exposure is not linked to cytotoxicity. Next day, colonoids were harvested from Matrigel using Cultrex Organoid Harvesting Solution, then resuspended in TrypLE enzyme solution (ThermoFisher), transferred to a 24-well plate and spun at at 37°C for 45 min in a spinoculator (Mixer HC; USA Scientific) to digest into single cells. Single cells were washed in of Advanced DMEM/F12, pelleted again, resuspended in of organoid expansion media and filtered with Flowmi cell strainers (SP Bel-Art). Cells were transported to the UNM Analytical and Translational Genomics Core in the UNM Comprehensive Cancer Center for counting and processing. Average processing time from colonoids to library preparation was hours, and the cells were highly viable ( ).
3
Intestinal Organoid Culture and Differentiation
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Intestinal organoids from paediatric patients were cultured in Matrigel (Corning). During organoid culture, the medium was replaced every 48–72 h. Organoids were passaged using mechanical disruption with a P1000 pipette and re-seeded in fresh growth-factor-reduced Matrigel (Corning). When comparing culture media, multiple wells were seeded from a single dissociated sample. The organoids were then allowed to grow for 5 days followed by 24 h treatment with recombinant human protein TNF (H8916, Sigma Aldrich) at 40 ng ml−1 or IFNγ (PHC4031, Life Technologies) at 20 ng ml−1. Organoids were in vitro differentiated for 4 days by culturing in a differentiation medium49 (link) and then collected for RNA extraction. Bright-field images were taken using an EVOS FL system (Life Technologies).
Processing for single-cell sequencing analysis was performed by removing the organoids from Matrigel at passage 3–4 using incubation with Cell Recovery Solution at 4 °C for 20 min, pelleting the cells, and re-suspending in TrypLE enzyme solution (Thermo Fisher) for incubation at 37 °C for 10 min. Cells were pelleted again and re-suspended in DMEM/F12.
Processing for single-cell sequencing analysis was performed by removing the organoids from Matrigel at passage 3–4 using incubation with Cell Recovery Solution at 4 °C for 20 min, pelleting the cells, and re-suspending in TrypLE enzyme solution (Thermo Fisher) for incubation at 37 °C for 10 min. Cells were pelleted again and re-suspended in DMEM/F12.
4
Fetal Intestinal Organoid Culture
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Fetal organoids were cultured according to a protocol by (Fordham et al., 2013) . During organoid culture, the media was replaced every 48-72 hours. For full media composition, see supplementary table 3. Organoids were passaged using mechanical disruption with a P1000 pipette and re-seeding in fresh growth-factor reduced Matrigel® (Corning). Processing for single-cell sequencing analysis was performed by removing the organoids from Matrigel using incubation with Cell Recovery Solution at 4 o C for 30 minutes, pelleting the cells, and resuspending in TrypLE enzyme solution (Thermo Fisher) for incubation at 37 o C for 15 mins. Cells were pelleted again and re-suspended in DMEM/F12. Cells from fetal ileum from embryos of 6 PCW were dissociated into single-cell suspension at passage 1 (~1 week) or passage 17 (~1 month) in culture and profiled using 10x Genomics single-cell transcriptomics. Brightfield images of organoids were taken using an EVOS Cell Imaging Systems microscope (Thermo Fisher).
5
Fetal Liver Cell Culture Protocol
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Fetal liver cell cultures were performed as previously described (75 (link)). Cultures were grown in 6 wells plates with William’s E Medium (Gibco) and epidermal growth factor 0.2 ug/ml and Cell Maintenance Supplement Pack (#CM4000, Thermo Fisher Scientific) providing 0.1 uM dexamethasone, 6.25 ug/ml human recombinant insulin, 6.25 ug/ml human transferrin, 6.25 ng/ml selenous acid,1.25 mg/ml bovine serum albumin, 5.35 ug/ml linoleic acid, 2 mM GlutaMAX and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Cells were harvested with TrypLE Enzyme solution (Life Technologies), stained, and analyzed by flow cytometry.
6
Hepatocyte Isolation and Culture
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CD235a− liver cells were plated in William's E Medium (Thermo Fisher Scientific, Grand Island, NY) with 5% FBS and Primary Hepatocyte Maintenance Supplements (#CM4000, Thermo Fisher Scientific) providing 0.1 µM dexamethasone, 6.25 µg ml−1 human recombinant insulin, 6.25 µg ml−1 human transferrin, 6.25 ng ml−1 selenous acid, 1.25 mg ml−1 bovine serum albumin, 5.35 µg ml−1 linoleic acid, 2 mM GlutaMAX and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Cultures were also supplemented with 10 ng ml−1 oncostatin M (OSM) and 10 ng ml−1 epidermal growth factor (EGF). Cells were grown on BioCoat Collagen I coated tissue culture plates (BD, Franklin Lakes, NJ) for 6 days at 37° C. Confluent cultures were harvested using TrypLE Enzyme solution (Life Technologies) and then stained for flow cytometric analysis or transplantation into mice. Some plates were fixed with 10% formalin in PBS and stained for immunofluorescence microscopy analysis.
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