PANC-1 and ASPC-1 cell lines were a gift from the Wilhelm Krek Lab (ETH Zurich, Switzerland). PDAC monolayer cell lines were established by culturing organoid cell suspensions on cell culture plates. Cells were grown in the following media supplemented with 50 U/mL penicillin, 50 U/L of streptomycin and 10% fetal bovine serum: DMEM (PANC-1) and RPMI 1640 (ASPC-1, PDAC Organoid derived monolayer cell lines). For monolayer screening cells were trypsinized and counted using Countess I Cell Counter (Invitrogen) 1’000 cells per well were added to a 384-well plate with the corresponding compounds of interest. The plate was incubated for 72 h and the readout performed using CellTiter-Glo®3D reagent according to the manufacturer’s protocol. Luminescence was measured by the BioTek plate reader Synergy H1 using Gen5 3.03. The raw luminescence data were filtered for outliers and converted to viability in [%] by normalizing to the DMSO controls. All drug screening experiments have been performed in at least two biological and two technical replicates.
Countess 1 cell counter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Countess I Cell Counter is a compact, automated cell counting device designed for quick and accurate cell enumeration. It utilizes a combination of advanced optics and image analysis algorithms to measure cell concentration and viability in a variety of sample types.
Automatically generated - may contain errors
4 protocols using countess 1 cell counter
1
Monolayer screening of pancreatic cancer cell lines
2
PDAC Cell Line Viability Assay
3
Cell Size Quantification Methods
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Changes in cell size were assessed by the measurement of the average cell diameter in microns with an automated cell counter (Countess 1 Cell Counter, Thermo Fisher Scientific, Waltham, MA, USA), and the relative differences in size based on the FSC-A parameter (forward scatter) by flow cytometry (FACSLyric, Becton Dickinson, Franklin Lakes, NJ, USA). Both methods enable the exclusion of debris and dead cells from the cell counts, eliminating the risk of their impact on the results. Dead cells (taking up trypan blue) were excluded from the analysis with an automated cell counter. In flow cytometry, dead cells were excluded based on FSC/SSC scattergram or high level FVS450 incorporation.
4
Human Erythroleukemia K562 Cell Culture
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The human erythroleukemia K562 cells were purchased from the American Type Culture Collection (ATTC) (Manassas, VA, USA) and cultured at 37 °C in a humidified atmosphere of 5% CO2 in air in RPMI 1640 medium (LONZA, Basel, Switzerland) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Waltham, MA, USA), 100 U/mL penicillin (Millipore Sigma, St. Louis, MO, USA), and 100 μg/mL streptomycin (Millipore Sigma, St. Louis, MO, USA). Cell proliferation and viability were determined with the trypan blue exclusion test and an automated cell counter (Countess 1 Cell Counter, Thermo Fisher Scientific, Waltham, MA, USA) each day of culture. All experiments were repeated at least three times. For each drug concentration, triplicate cultures were used. Vehicle controls of 7.3 mM NaOH for hemin and 0.05% DMSO for everolimus were run in each experiment.
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