Nuclei were isolated from liquid nitrogen flash frozen PDOX tumors [92 (link)]. Samples were minced in DAPI buffer [10 μg/ml DAPI in 146 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.2% IPEGAL]. Nuclei were disaggregated subsequently with 20G and 25G needles and filtered through a 50 μm and a 30 μm mesh. Tumor nuclei were stained with the human-specific anti-Lamin A/C-PE antibody (Supplementary Table 2, online resource). Optionally, PDOX-derived single cell tumor cells and cell lines were stained with IR-LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen; 1 µg/ml) and fixed with cold 80% ethanol. PBMCs were added to each sample as internal diploid control. Flow analysis was carried out with AriaTMSORP or CantoTM flow cytometers (BD Biosystems). DNA content was analyzed with the FlowJo software.
Golebiewska A., Hau A.C., Oudin A., Stieber D., Yabo Y.A., Baus V., Barthelemy V., Klein E., Bougnaud S., Keunen O., Wantz M., Michelucci A., Neirinckx V., Muller A., Kaoma T., Nazarov P.V., Azuaje F., De Falco A., Flies B., Richart L., Poovathingal S., Arns T., Grzyb K., Mock A., Herold-Mende C., Steino A., Brown D., May P., Miletic H., Malta T.M., Noushmehr H., Kwon Y.J., Jahn W., Klink B., Tanner G., Stead L.F., Mittelbronn M., Skupin A., Hertel F., Bjerkvig R, & Niclou S.P. (2020). Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology. Acta Neuropathologica, 140(6), 919-949.
Other organizations :
Laboratoire National de Santé, University of Luxembourg, Centre Hospitalier de Luxembourg, German Cancer Research Center, Heidelberg University, University Hospital Heidelberg, National Center for Tumor Diseases, Delmar (Canada), Haukeland University Hospital, Henry Ford Health System, German Cancer Society, University Hospital Carl Gustav Carus, TU Dresden, St James's University Hospital
PDOX tumor samples were flash frozen in liquid nitrogen
dependent variables
DNA content of tumor nuclei
Viability of PDOX-derived single cell tumor cells and cell lines
control variables
DAPI buffer composition (10 μg/ml DAPI, 146 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.2% IPEGAL)
Needle size used for nuclei disaggregation (20G and 25G)
Mesh sizes used for filtration (50 μm and 30 μm)
Human-specific anti-Lamin A/C-PE antibody used for tumor nuclei staining
IR-LIVE/DEAD® Fixable Dead Cell Stains used for PDOX-derived single cell tumor cells and cell lines
80% cold ethanol used for fixation of PDOX-derived single cell tumor cells and cell lines
PBMCs added as internal diploid control
controls
Positive control: PBMCs added as internal diploid control
Negative control: Not explicitly mentioned
Annotations
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