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Experiment Overview

Repository ID: FR-FCM-ZZS3 Experiment name: multiparametric analysis of complex cellular response MIFlowCyt score: 67.91%
Primary researcher: Šárka Šimečková PI/manager: Karel Soucek Uploaded by: Šárka Šimečková
Experiment dates: 2014-01-01 - 2015-08-31 Dataset uploaded: Jun 2016 Last updated: Dec 2017
Keywords: [flow cytometry] [APOPTOSIS] [multiparametric analysis] [DNA damage] [proliferation] [click chemistry] [cancer stem cell markers] Manuscripts: [29220555] Cytalogo
Organizations: Institute of Biophysics AS CR, v.v.i., Department of Cytokinetics, Brno, (Czech Republic)
Purpose: To introduce a protocol for a simultaneous flow cytometric detection of two CD surface antigens, DNA synthesis and quantification, histone H2AX phosphorylation (marker for DNA damage), cleaved PARP (marker for apoptosis) and viability on representative panel of cell lines derived from human and mouse.
Conclusion: We introduced protocol for a simultaneous flow cytometric detection of two CD surface antigens, DNA synthesis and quantification, histone H2AX phosphorylation (marker for DNA damage), cleaved PARP (marker for apoptosis) and viability on representative panel of cell lines derived from human and mouse. This method is useful tool for complex analysis of cells.
Comments: None
Funding: Not disclosed
Quality control: For compensation setup were used anti-rat and anti-hamster Ig kappa/negative control compensation beads (BD Biosciences), SpheroTM Biotin Polystyrene Particles (Spherotech, Lake Forest, IL, USA) labeled with antibodies (Table S2) in appropriate dilution. ArcTM Amine Reactive Compensation Bead Kit beads (Thermo Fisher Scientific) were stained with LIVE/DEAD® Fixable fluorescent reactive dye kit. As compensation controls for DNA stain, fixed and permeabilized cells with/without appropriately diluted DNA probe were used. Compensations were calculated using FACSDiva software (Version 6.1.3; BD Biosciences)


Experiment variables

Sample Type
· lymphoma cell line 150717_SU-DHL-4 ISO Ctrl.fcs · 150717_SU-DHL-4 ISO MG132.fcs · 150717_SU-DHL-4 ISO MLN.fcs · 150717_SU-DHL-4 SP Ctrl.fcs · 150717_SU-DHL-4 SP MG132.fcs · 150717_SU-DHL-4 SP MLN.fcs
· prostate cancer cell line (human) 150717_DU145 Ctrl.fcs · 150717_DU145 ISO Ctrl.fcs · 150717_DU145 MLN ISO.fcs · 150717_DU145 MLN SP.fcs · 150717_DU145 TRAIL ISO.fcs · 150717_DU145 TRAIL SP.fcs
· CRC cell line 150618_HCT116 ISO MLN.fcs · 150618_HCT116 ISO Trail.fcs · 150618_HCT116 ISO ctrl.fcs · 150618_HCT116 SP MLN.fcs · 150618_HCT116 SP Trail.fcs · 150618_HCT116 SP ctrl.fcs
· prostate cancer cell line (mouse) 150618_cE2 ISO MG.fcs · 150618_cE2 ISO MLN.fcs · 150618_cE2 ISO ctrl.fcs · 150618_cE2 SP MG.fcs · 150618_cE2 SP MLN.fcs · 150618_cE2 SP ctrl.fcs

Conditions
· MLN-4924 treatment 150618_HCT116 ISO MLN.fcs · 150618_HCT116 SP MLN.fcs · 150618_cE2 ISO MLN.fcs · 150618_cE2 SP MLN.fcs · 150717_DU145 MLN ISO.fcs · 150717_DU145 MLN SP.fcs · 150717_SU-DHL-4 ISO MLN.fcs · 150717_SU-DHL-4 SP MLN.fcs
· MG-132 treatment 150618_cE2 ISO MG.fcs · 150618_cE2 SP MG.fcs · 150717_SU-DHL-4 ISO MG132.fcs · 150717_SU-DHL-4 SP MG132.fcs
· TRAIL treatment 150618_HCT116 ISO Trail.fcs · 150618_HCT116 SP Trail.fcs · 150717_DU145 TRAIL ISO.fcs · 150717_DU145 TRAIL SP.fcs
· DMSO - control 150618_HCT116 ISO ctrl.fcs · 150618_HCT116 SP ctrl.fcs · 150618_cE2 ISO ctrl.fcs · 150618_cE2 SP ctrl.fcs · 150717_DU145 Ctrl.fcs · 150717_DU145 ISO Ctrl.fcs · 150717_SU-DHL-4 ISO Ctrl.fcs · 150717_SU-DHL-4 SP Ctrl.fcs

Doses
· MG132 1 uM 150618_cE2 ISO MG.fcs · 150618_cE2 SP MG.fcs · 150717_SU-DHL-4 ISO MG132.fcs · 150717_SU-DHL-4 SP MG132.fcs
· MLN-4924 1 uM 150717_SU-DHL-4 ISO MLN.fcs · 150717_SU-DHL-4 SP MLN.fcs
· DMSO 150618_HCT116 ISO ctrl.fcs · 150618_HCT116 SP ctrl.fcs · 150618_cE2 ISO ctrl.fcs · 150618_cE2 SP ctrl.fcs · 150717_DU145 Ctrl.fcs · 150717_DU145 ISO Ctrl.fcs · 150717_SU-DHL-4 ISO Ctrl.fcs · 150717_SU-DHL-4 SP Ctrl.fcs
· TRAIL 50 ng/ml 150618_HCT116 ISO Trail.fcs · 150618_HCT116 SP Trail.fcs · 150717_DU145 TRAIL ISO.fcs · 150717_DU145 TRAIL SP.fcs
· MLN-4924 5 uM 150618_cE2 ISO MLN.fcs · 150618_cE2 SP MLN.fcs
· MLN-4924 0.33 uM 150618_HCT116 ISO MLN.fcs · 150618_HCT116 SP MLN.fcs · 150717_DU145 MLN ISO.fcs · 150717_DU145 MLN SP.fcs

Timepoints
· 24 hours treatment (DMSO/MLN-4924/MG-132) 150618_HCT116 ISO MLN.fcs · 150618_HCT116 ISO ctrl.fcs · 150618_HCT116 SP MLN.fcs · 150618_HCT116 SP ctrl.fcs · 150618_cE2 ISO MG.fcs · 150618_cE2 ISO MLN.fcs · 150618_cE2 ISO ctrl.fcs · 150618_cE2 SP MG.fcs · 150618_cE2 SP MLN.fcs · 150618_cE2 SP ctrl.fcs · 150717_DU145 Ctrl.fcs · 150717_DU145 ISO Ctrl.fcs · 150717_DU145 MLN ISO.fcs · 150717_DU145 MLN SP.fcs · 150717_SU-DHL-4 ISO Ctrl.fcs · 150717_SU-DHL-4 ISO MG132.fcs · 150717_SU-DHL-4 ISO MLN.fcs · 150717_SU-DHL-4 SP Ctrl.fcs · 150717_SU-DHL-4 SP MG132.fcs · 150717_SU-DHL-4 SP MLN.fcs
· 2 hours treatment (TRAIL) 150618_HCT116 ISO Trail.fcs · 150618_HCT116 SP Trail.fcs · 150717_DU145 TRAIL ISO.fcs · 150717_DU145 TRAIL SP.fcs

Individuals
· Prominin-1_optimization Fix ISO.fcs · Fix SP.fcs · L_D ISO.fcs · L_D SP.fcs · Perm ISO.fcs · Perm SP.fcs · click ISO.fcs · click SP.fcs
· cE2_results 150618_cE2 ISO MG.fcs · 150618_cE2 ISO MLN.fcs · 150618_cE2 ISO ctrl.fcs · 150618_cE2 SP MG.fcs · 150618_cE2 SP MLN.fcs · 150618_cE2 SP ctrl.fcs
· HCT116_results 150618_HCT116 ISO MLN.fcs · 150618_HCT116 ISO Trail.fcs · 150618_HCT116 ISO ctrl.fcs · 150618_HCT116 SP MLN.fcs · 150618_HCT116 SP Trail.fcs · 150618_HCT116 SP ctrl.fcs
· DU145_results 150717_DU145 Ctrl.fcs · 150717_DU145 ISO Ctrl.fcs · 150717_DU145 MLN ISO.fcs · 150717_DU145 MLN SP.fcs · 150717_DU145 TRAIL ISO.fcs · 150717_DU145 TRAIL SP.fcs
· SU-DHL-4_results 150717_SU-DHL-4 ISO Ctrl.fcs · 150717_SU-DHL-4 ISO MG132.fcs · 150717_SU-DHL-4 ISO MLN.fcs · 150717_SU-DHL-4 SP Ctrl.fcs · 150717_SU-DHL-4 SP MG132.fcs · 150717_SU-DHL-4 SP MLN.fcs
· DAPI_vs_PO-PRO-1 150624_cE2 ISO ctrl_POPRO1_BV650.fcs · 150624_cE2 SP ctrl_POPO1_BV650.fcs · ISO_cE2_PE.fcs · SP_cE2_Trop2_PE.fcs · 150526_cE2_Ctrl ISO.fcs · 150526_cE2_Ctrl ISO_noDAPI.fcs · 150526_cE2_Ctrl SP_Click.fcs
· CC3_PARP cE2_MG132 CASP3.fcs · cE2_MG132 ISO.fcs · cE2_MG132 PARP.fcs · cE2_ctrl CASP3.fcs · cE2_ctrl ISO.fcs · cE2_ctrl PARP.fcs
· Permeabilization reagents comparison 10.fcs · 12.fcs · 14.fcs · 16.fcs · 2.fcs · 3.fcs · 6.fcs · 8.fcs

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