Karyotyping Organoids via Colcemid

Karyotyping was performed as previously described 24 (link). Briefly, cultures were incubated with 0.1ug/ml Karyomax Colcemid (Gibco). After 24 hours, organoids were harvested and dissociated using TrypLE (Gibco). Cells were incubated with KCL 0.0075M hypotonic solution for 10 min, fixed in methanol:acetic acid (3:1) and dropped on a microscope slide for visualization. Nuclei were mounted and stained using Vectashield with DAPI (Vector Labs). A minimum of 15 metaphases per sample were counted.

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Broutier L., Mastrogiovanni G., Verstegen M.M., Francies H.E., Gavarró L.M., Bradshaw C.R., Allen G.E., Arnes-Benito R., Sidorova O., Gaspersz M.P., Georgakopoulos N., Koo B.K., Dietmann S., Davies S.E., Praseedom R.K., Lieshout R., IJzermans J.N., Wigmore S.J., Saeb-Parsy K., Garnett M.J., van der Laan L.J, & Huch M. (2017). Human Primary Liver Cancer -derived Organoid Cultures for disease modelling and drug screening. Nature medicine, 23(12), 1424-1435.

Publication 2017

Karyomax Colcemid TrypLE Vectashield with DAPI

Acetic acid Cells Colcemid Dapi Hypotonic solution Metaphases Methanol Microscope Nuclei Organoids Vector

Corresponding Organization : University of Cambridge

Other organizations : Wellcome/Cancer Research UK Gurdon Institute, Wellcome Trust, Erasmus MC, Wellcome Sanger Institute, Wellcome/MRC Cambridge Stem Cell Institute, Medical Research Council, Cambridge University Hospitals NHS Foundation Trust, Edinburgh Royal Infirmary

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Variable analysis

independent variables

Karyotyping protocol

dependent variables

Metaphase counts

control variables

Incubation with 0.1ug/ml Karyomax Colcemid (Gibco) for 24 hours
Incubation with KCL 0.0075M hypotonic solution for 10 min
Fixation in methanol:acetic acid (3:1)
Mounting and staining using Vectashield with DAPI (Vector Labs)

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