Computational protocol: A new mild hyperthermia device to treat vascular involvement in cancer surgery

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Protocol publication

[…] Cytotoxicity was quantified using DNA-binding DRAQ7 (Biostatus Ltd, UK) staining, which is a far-red fluorescent dye. Cells were seeded at a concentration of 100,000 cells per well in Greiner Bio One Cellstar® tissue culture 6-well plate for 24 h. Plates were then placed into a water bath and heated to the given thermal dose. An optical probe was inserted into a cell free well that contained 2 ml of media so that accurate thermometry of the plate bottom could be achieved. After 10 mins of water bath hyperthermia, 3 µM concentration of DRAQ7 was placed in the cell medium before being gently pipetted to ensure thorough mixing. High throughput microscopy was performed within 10 min of adding the DRAQ7 using the ImageXpress Micro XL microscope (GE Healthcare, USA). Images were obtained which included 10,000 cells at 0 h and 24 h time points in triplicate for statistically robust findings. Cells were imaged at 20x magnification in two wavelengths: the DRAQ7 (Cy5, Absorbance 600 nm emission 697 nm) and the bright-field wavelengths. At the end of the time-lapse experiment the cells were placed in 52 °C water for 10 minutes to induce 100% cell death before an additional set of images was collected, from which a measure of the total number of cells in each well was obtained. Cell death was quantified by the automated measure of the DRAQ7 signals at 0 and 24 h time points using MetaXpress 5.3.0.5 and MetaXpress PowerCore 1.3.0.3 software (Molecular Devices, USA). [...] Cells were seeded at a concentration of 100,000 cells per well in Greiner Bio One Cellstar® tissue culture 6-well plate for 24 h. Plates were then placed into a water bath and heated to the target thermal dose. An optical probe was inserted into a cell free well that contained 2 ml of media so that accurate thermometry of the plate bottom could be achieved. After 10 mins of water bath hyperthermia, the media was collected and the cells were trypsanised before being added to the supernatant media and spun down. For the tumor sphere-forming assay the cells were reseeded in six-well ultra-low attachment plates (Corning) with 2 mL of serum-free mammosphere medium. Fresh mammosphere medium was added to the wells every 3 days. The number of viable PDAC spheres at 14 days were imaged using ImageXpress Micro XL microscope (GE Healthcare, USA) and counted using MetaXpress 5.3.0.5 software (Molecular Devices, USA).For the surface marker expression analysis, APC-labeled mouse anti-human CD44 (clone G44–26) and FITC-labeled mouse anti-human CD24 (clone mL5) were both purchased from BD Pharmingen (San Diego, CA). 7-AAD Viability Staining Solution was purchased from eBioscience (San Diego, CA). PANC-1 cells were removed from flasks using Enzyme Free Cell Dissociation Buffer (ThermoFisher) and rinsed in FACs buffer (PBS + 2% FBS) prior to staining. Cells were then stained for surface antigens according to the antibody manufacturer’s instructions. Following the last rinse to remove any free antibody, cells were suspended in 200 µL PBS and 5 µL 7-AAD viability stain was added 5–15 minutes prior to analysis on a Attune NXT flow cytometer (ThermoFisher). Analysis was performed using FlowJo version 10. Single color and unstained controls were used for proper gating and compensation. […]

Pipeline specifications

Software tools MetaXpress, FlowJo
Application Flow cytometry