Experiment
Overview
Purpose/Goal/Hypothesis – Assessment of apoptosis of IMR-32 cells upon 30 h treatment with GSK3 inhibitors
(10μM AZAkenpaullone, 10μM BIOacetoxime, 2 mM LiCl) and 10μM Wnt agonist 1.
Experiment Variables –
Samples were obtained and prepared under same experimental conditions using cells with low passage number;
Samples were analysed without fixation;
Samples for cells treated with 10μM BIOacetoxime display a shift in fluorescence compared to controls intensity
due to auto-fluorescence of this compound (not yet reported in the literature);
Activated caspase assay was performed using CaspACE FITC-VAD-FMK In Situ Marker in combination with PI;
Membrane integrity assay was performed using Yo-Pro-1 dye only.
Conclusions – Analysis revealed that apoptosis is the major mechanism for cell death in cell treated with
AZAkenpaullone, LiCl and Wnt Agonist 1 and necrosis in cells treated with BIOacetoxime.
Quality Control –
Experiments were performed in technical triplicates;
Control treatments were performed with 0.1% DMSO for all compounds, except for LiCl where 28 mM NaCl was
used;
Weekly instrument calibration using 6- / 8-peak bead standards recommended by manufacturer BD Biosciences.
Flow Sample
(Specimen)
Material – IMR32 neuroblastoma cell line
Source/Organism/Location – Metastatic neuroblastoma tissue; Homo sapiens
Treatment – 30 hours treatment, followed by cell harvesting, washing with PBS and staining of samples
according to manufacturers’ instructions.
Reagent/Analyte/Detector/Reporter –
PI (Propidium Iodide) for the assessment of membrane integrity and cell viability; excited by the 488nm laser:
emission collected using the default 675 LP filter in FL3;
Yo-Pro-1(Invitrogen) for the assessment of apoptosis/ disrupted membrane integrity;
CaspACE FITC-VAD-FMK In Situ Marker (Promega) for the assessment of apoptosis by in situ labelling of
activated caspases.
Yo-Pro-1 and CaspACE-FITC were measured independently, as both of them are excited with the 488nm laser,
emission collected using the default filter, 530/30 in FL1.
Data Analysis
List-mode Data – c6
Compensation – No compensation
Gating – Initial gating in FSC-A/SSC-A removing cell debris;
Gating in 488: 530/30: CaspFITC-A/488: 670 LP: PI-A for PI- Casp-, PI- Casp+, PI+ Casp+, PI+ Casp- ;
Gating in in 488: 530/30: YoPro1-A /488: 670 LP: PI-A for PI- Yo-Pro1-, PI- Yo-Pro1+, PI+ Yo-Pro1+, PI+ YoPro1-;
Gates were set up using single stain controls. Some minor adjustments on the gates should be done due to an
excess of the markers;
BIO treated samples were gated separately due to their readout shift when compared to control samples and other
treatments. The shift is a consequence of the auto-fluorescence for this compound which has not yet been reported
in the literature. Reliable gating and interpretation of results in Yo-Pro-1 staining assay was not possible for BIO
treated samples because of the spectral overlap of BIO with PI.
Reliable gating not possible for BIOacetoxime treated cells due to spectral overlap of the drug with PI;
Separate CaspPI gating for samples treated with 10μM BIOacetoxime due to fluorescence shift when compared to
control samples. Gates for this treatment were based on their own single label controls and unstained controls.
Descriptive statistics – Average value of each technical triplicate and standard deviation
Instrument
Details
Instrument Identification -BD Accuri C6 flow cytometer (BD Biosciences)
Fluidics Configuration - Low-pressure peristaltic pumping system
Optical Configuration – Standard 3-blue 1-red configuration with detectors: FL1 - filter 530/30, FL2 – 585/40,
FL3 – 670 LP, FL4 – 675/25
Electronic Configuration – Default instrument dynamic range