Human neoplastic organoid cultures were established from resected PDAC specimens. Specimens from pancreatic resections were digested enzymatically with collagenase/dispase dissociation and then plated in Matrigel to generate pancreas organoid cultures, in human complete medium [25 (link)]. PDAC organoids were then maintained in human complete medium. For the 50% of cell growth inhibition (IC50) analysis, organoids were dissociated into single cells by first triturating them in media through a fire-polished glass pipette, and then by enzymatic dissociation with 2 mg/ml dispase dissolved in TrypLE (Life Technologies). Cells were counted and diluted to 10 cells/μl in a mixture of complete media, Rho Kinase inhibitor Y-27632 (10.5 μM final concentration, Sigma) and Growth factor-reduced Matrigel (GFR-Matrigel, 10% final concentration). 100 μl of this mixture (1000 cells/well) was plated in 96-well plates (Nunc), whose wells had been previously coated with a bed of GFR-Matrigel to prevent attachment of the cells to the bottom of the plate. Cell viability in response to the different drugs was measured using the CellTiter-Glo assay (Promega Corporation) and Synergy 4 reader (Biotek).
Synergy 4 reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 4 reader is a multi-mode microplate reader designed for a wide range of applications. It is capable of absorbance, fluorescence, and luminescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Rho kinase inhibitor y 27632, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Dabrafenib, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by Thomas Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
Gen5 1, by Agilent Technologies (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
6 protocols using synergy 4 reader
1
Establishment and Evaluation of PDAC Organoids
2
Dabrafenib Dose-Response Assay
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Cells were plated at a concentration of 5000 cells onto 96 well plates and incubated at 1% FBS (Denville Scientific). Dabrafenib was purchased from Selleckchem. Cells were treated with Dabrafenib at concentrations of 0.1–100 nM. At 48 h fresh drug was applied to cells. After 72 h cells were brought up to room temperature and 100 µL of CellTiter-Glo (Promega) reagent was added directly to each well. Plates were incubated on a shaker for 5 min and luminescence was measured on a Synergy 4 reader (Biotek). Luminescence readings were normalized to and shown as a relative percentage of DMSO control readings.
3
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis by flow cytometry, ethanol-fixed cells were stained with 5 μg/ml propidium iodide (PI) and 250 μg DNase-free RNAse. A total of 50,000 cells were acquired in the PI gate by using a BD Accuri™ C6 flow cytometer and the C6 (version 1.0.264.21) software (BD Biosciences).
To measure bromodeoxyuridine (BrDU) incorporation, the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics GmbH, Mannheim, Germany) was used following the manufacturer's instructions. Data were acquired using a BioTek Synergy 4 reader with Gen5 1.05 software (both from BioTek Instruments, Inc., Winooski, VT, USA).
To measure bromodeoxyuridine (BrDU) incorporation, the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics GmbH, Mannheim, Germany) was used following the manufacturer's instructions. Data were acquired using a BioTek Synergy 4 reader with Gen5 1.05 software (both from BioTek Instruments, Inc., Winooski, VT, USA).
4
CSF Collection and sPDGFRβ Quantification
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Mouse CSF was collected as described in Lim’s protocol65 (link). Briefly, under anesthesia (1% isoflurane inhalation), the cisterna magna was exposed by removing nearby muscles. Then, a clean glass capillary was punctured into the cisterna magna, and CSF was automatically drawn into the capillary tube. We obtained 8–15 µl CSF per mouse. To avoid the possible sPDGFRβ concentration fluctuations due to circadian time, we always collect CSF samples at ZT05-06. Then, the standard sandwich ELISA assay was performed according to the manufacturer's protocol (Abcam, Cambridge, MA). The plate was read immediately on the BioTek Synergy 4 reader. Soluble PDGFRβ (sPDGFRβ) concentrations were calculated using the samples’ readings and the linear standard curve equation. The results were multiplied by the dilution factor to arrive at the final concentration in the original CSF samples.
5
Caco-2 Cytotoxicity Evaluation of Grape Extracts
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Human intestinal epithelial cells (Caco-2) were grown as monolayer in 75 cm 2 flasks, incubated at 37 °C in 100% humidity and 5% CO2. Dulbecco's Modified Eagle Medium with high glucose and L-glutamine, supplemented with 20% foetal bovine serum, 10% penicillin and streptomycin and 1% fungizone, was used as growth medium.
The cells were seeded into 96-well plates at a density of 7.5×10 3 cells/well. After 24 h of incubation, the cells were treated with the grape ethanol/propylene glycol extract in aqueous solution or loaded in PEVs and nutriosomes properly diluted with DMEM to achieve the desired extract concentrations (6, 12, 60, 120 μg/ml). After 48 h, MTT [3(4,5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide] (100 μl, 0.5 mg/ml final concentration) was added to each well. After 3 h, the formed formazan crystals were dissolved with DMSO, and the absorbance was measured at 570 nm with a microplate reader (Synergy 4 Reader, BioTek Instruments, AHSI S.p.A, Bernareggio, Italy). The experiment was repeated at least three times, each time in triplicate.
The cells were seeded into 96-well plates at a density of 7.5×10 3 cells/well. After 24 h of incubation, the cells were treated with the grape ethanol/propylene glycol extract in aqueous solution or loaded in PEVs and nutriosomes properly diluted with DMEM to achieve the desired extract concentrations (6, 12, 60, 120 μg/ml). After 48 h, MTT [3(4,5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide] (100 μl, 0.5 mg/ml final concentration) was added to each well. After 3 h, the formed formazan crystals were dissolved with DMSO, and the absorbance was measured at 570 nm with a microplate reader (Synergy 4 Reader, BioTek Instruments, AHSI S.p.A, Bernareggio, Italy). The experiment was repeated at least three times, each time in triplicate.
6
Multiparametric Cytotoxicity Assay
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After 24 h treatment, the exposed cells were rinsed with PBS (pH 7.2) and incubated in serum-free culture media containing 5% v/v Alamar Blue® and 4 µM CFDA-AM. After a 30 min incubation, the fluorescence was measured using microplate reader Synergy 4 Reader (BioTek, Winooski, VT, USA) at 485/520 nm excitation/emission for CFDA and 530/590 nm excitation/emission for Alamar Blue®. The cells were then rinsed with PBS and incubated for 2 h with 50 µg/mL of neutral red dissolved in serum-free culture media and washed again with PBS. Accumulated neutral red was extracted with 50% (v/v) ethanol-1% (v/v) acetic acid and quantified spectrophotometrically (Synergy 4) at 540 nm with 690 nm reference wavelength. Fluorescence and absorbance readings from the assay blank wells without cells were subtracted from the experimental wells before data analysis.
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