Human pancreas, stomach and colon organoids were cultured in the previously described standard organoid culture media (Bartfeld et al., 2015 (link); Boj et al., 2015 (link); Sato et al., 2011 (link)). Organoids were collected, pelleted, resuspended in TrypLE Express 1X (Gibco #12604013) and incubated for 5 minutes at 37°C in order to dissociate the culture into a single cell suspension. DAPI was added to the suspension and living cells were FACS sorted (BD FACSARIA II) into collection tubes. 50 cells were seeded per well into 96 well plates and standard media were added containing 10 mM ROCK inhibitor Y-27632 (Abmole, M1817) and either Wnt3a CM (50%) (Homemade) or 0.5 nM NGS Wnt. Media was refreshed every 4 days. Organoids were incubated for 2 weeks at 37°C and 5% CO2 after which organoid growth was assessed by means of organoid counting and cell viability quantification using CellTiter-Glo® Luminescent Cell Viability Assay (Promega # G7572). 50 μL of CellTiter-Glo® solution was added directly to the organoids in 100 μL medium. Luminescence was measured using Spark® multimode microplate reader (TECAN).
Tryple express 1x
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
TrypLE Express (1x) is a cell dissociation reagent used for the detachment and dissociation of adherent cells from cell culture vessels. It is a ready-to-use solution that can be directly applied to cell cultures.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Facsaria 2, by BD (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Kojic acid, by Merck Group (1 mentions)
Copper sulfate, by Merck Group (1 mentions)
Phosphate buffer saline (pbs), by Thermo Fisher Scientific (1 mentions)
Pyrocatechol violet, by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Facsaria 3 ow cytometer, by BD (1 mentions)
Facsverse ow cytometer, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
14 protocols using tryple express 1x
1
Organoid Culture and Analysis
2
Blue Light Induces Caspase Activation
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Transfected HeLa or HEK293T cells were either exposed to blue light (30 μmol m−2 s−1) or kept in the dark for the indicated time. Then, the cells were dissociated from the dishes with TrypLE™ Express (1x) (Gibco, 12605-028) at 37 °C for 5 min. After centrifugation at 800×g for 5 min, the supernatant was discarded. The cell pellets were lysed with Pierce IP Lysis Buffer (87787, Pierce) supplemented with 1× EDTA-free Protease Inhibitor Cocktail Tablets (4693159001, Roche) and incubated on ice for 15 min. The mixtures were centrifuged at 14,000×g for 10 min at 4 ℃ to remove cell debris. The supernatants were boiled with 4× Loading buffer for 10 min. Then, the samples were detected by western blot and probed with anti-Caspase8 (Abcam, ab32397), anti-Caspase3 (MBL, M097-3), and anti-Actin (MBL, M177-3). The Western blot band intensities were analyzed with ImageJ software, and all the band intensities were normalized to actin.
3
Isolation and Characterization of Thermoactinomyces Antibioticus Enzyme TR
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TR was originally isolated from Thermoactinomyces antibioticus based on the method reported previously by Craveri et al. [23] . TR was obtained through Dr. Francis Johnson (Department of Chemistry, Stony Brook). Kojic acid (KA), L-ascorbic acid (AA), alpha-glucosidase enzyme (Baker's yeast), 4-Nitrophenyl α-d-glucopyranoside substrate (pNG), mushroom tyrosinase enzyme (T3824), L-DOPA (3,4-Dihydroxy-l-phenylalanine), Pyrocatechol violet (PV) and copper sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate monobasic (monohydrate, ACS Reagent; JT Baker, NJ, USA), TrypLE™ Express (1X), and Phosphate buffer saline (PBS) were procured from Gibco (Thermo Fisher Scientific, MA, USA). 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagents were procured from Molecular Probes (Eugene, OR, USA).
4
Isolation of Corneal Endothelial Cells
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HCEnCs were isolated from research grade donor corneas using a peel-and-digest method similar to previously published methods, [7 (link), 13 (link)–15 (link)] with limited modifications. Firstly, the corneas [n = 30] were washed in sterile PBS and Descemet's membrane with endothelium was dissected with a fine forceps, similar to the stripping technique used for Descemet's membrane endothelial keratoplasty (DMEK). Secondly, the excised pieces were incubated in 2 mg/mL collagenase type 1 (Thermo Fisher Scientific, Rochester, NY, USA) solution for 2-3 hours at 31°C, 5% CO2. Once Descemet's membrane was digested, the solution was centrifuged for 5 minutes at 1000 rpm. The supernatant was removed, and the cells were resuspended in TrypLE Express (1x) for 10 minutes at 37°C, (Life Technologies, Monza, Italy) to obtain single cell suspension suitable for seeding. An overview of the performed experiments can be seen in Figure 1 .
5
Tissue Dissociation and Cell Isolation
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The central and peripheral tissue pieces were incubated in 2 mg/mL collagenase type 1 diluted in human endothelium-SFM supplemented with 5% FBS and 1% PenStrep (Thermo Fisher Scientific, Rochester, NY, USA) solution for 2 hours at 31°C and 5% CO2. The cells collected with collagenase were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed, and the cells were resuspended with TrypLE express (1X) (Life Technologies, Monza, Italy) for 10 minutes at 37°C to obtain single cells. The cells were resuspended with 200 μL of the cell culture media after counting them using a haemocytometer slide.
6
Single-cell isolation from zebrafish embryos
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Embryos were collected at the stage of interest in 20 ml E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) in a glass beaker. 500μl 10mg/ml pronase was added to remove the chorions. The chorions were washed away with E3 and the embryos were taken up in 1 mL TrypLE Express (1x) (Life Technologies) and dissociated with the help of a syringe. The samples were added to 6 mL E3, 5 μL 1M EDTA and 0.5 mL FCS was added. Samples were centrifuged for 5 minutes at 1000g. Supernatant was removed and cells were taken up in PBS. Cells were pipetted through a cell strainer and 10 μL Dnase was added. Prior to sorting, DAPI was added. Single GFP-positive cells were sorted using a BD FACSAria III (Becton Dickinson) with an 85 μm nozzle in 96 well plates containing 150 μL TRIzol Reagent (ThermoFisher).
7
Analyzing Platelet-Cell Interactions by Flow Cytometry
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Flow cytometry experiments were performed using 24-wells plates and 500μl of platelets suspension. Experiments were run in triplicates and collected at different time points. After co-culture, platelets were harvested from cell media after centrifugation at 105 x g for 15 min to eliminate cell fragments. Cells were washed twice with PBS 1X to remove any remaining platelets and cell colonies were dissociated with Tryple Express 1X (Life Technologies). Cells and platelets suspensions were xed with 3.7% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min at room temperature.
For the analysis of cell activation, platelets were incubated with mouse anti-human PAC- Between primary and secondary antibody incubations, cells and platelets were washed with FACS Buffer (PBS 1X, 5% FBS, EDTA 2Mm) and PBS-EDTA 2mM, respectively.
Both cells and platelets were analyzed in the BD FACSVerse™ ow cytometer equipped with three lasers: violet (405nm), blue (488nm) and red (633nm) (BD Bioscience) using BD FACSuite™ software (BD Bioscience) for acquisition or by FACS ARIA III™ ow cytometer equipped with four lasers: violet (405nm), blue (488nm), yellow/green (531nm) and red (633nm) (BD Bioscience) using BD FACSDiva™ software (BD Bioscience) for acquisition and FlowJo™ for analysis (FlowJo, LLC-BD Bioscience). Flow cytometry gating strategy is described in Fig. S2.
For the analysis of cell activation, platelets were incubated with mouse anti-human PAC- Between primary and secondary antibody incubations, cells and platelets were washed with FACS Buffer (PBS 1X, 5% FBS, EDTA 2Mm) and PBS-EDTA 2mM, respectively.
Both cells and platelets were analyzed in the BD FACSVerse™ ow cytometer equipped with three lasers: violet (405nm), blue (488nm) and red (633nm) (BD Bioscience) using BD FACSuite™ software (BD Bioscience) for acquisition or by FACS ARIA III™ ow cytometer equipped with four lasers: violet (405nm), blue (488nm), yellow/green (531nm) and red (633nm) (BD Bioscience) using BD FACSDiva™ software (BD Bioscience) for acquisition and FlowJo™ for analysis (FlowJo, LLC-BD Bioscience). Flow cytometry gating strategy is described in Fig. S2.
8
Etoposide-Induced DNA Damage in iPSCs
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iPS cells were cultured in Essential 8TM medium (ThermoFisher Scientific, A1517001) on plates coated with human recombinant Laminin-521 (LN-521, 1:20 in phosphate-buffered saline (PBS), Biolamina, LN521-03). Passaging was performed using TrypLE Express 1X (ThermoFisher Scientific, 12604013). Cells were seeded in media supplemented with 10 μM Y27632 Rho-kinase inhibitor (ROCKi, Millipore, SCM075). To induce DNA damage, cells were incubated with 0.5 μM Etoposide (Merck, E1383) for 3 h before harvest.
9
Calu-3 Cell Culture Protocol
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Calu-3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (1:1) GlutaMAX™ (ThermoFisher Scientific) supplemented with 10% FBS, 1% NEAA, 1% sodium pyruvate, 1% penicillin-streptomycin and maintained at 37°C, 5% CO2 and passaged with TrypLE™ Express 1X (ThermoFisher Scientific) when ~80% confluent. All experimental work was performed on cells within the range of 20–30 passages.
10
Calu-3 Cell Culture Protocol
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Calu-3 cells were cultured in DMEM/F12 (1:1) GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS, 1% NEAA, 1% sodium pyruvate, 1% penicillin-streptomycin, and maintained at 37°C, 5% CO2, and passaged with TrypLE Express 1X (Thermo Fisher Scientific) when ∼80% confluent. All experimental work was performed on cells within the range of 20–30 passages.
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