Tumor tissues were dissected from patients, ≥18 years of age with primary GBM tumors, during surgery in the Neurosurgery Department of The Second Affiliated Hospital of Anhui Medical University (Hefei, China) and collected in sterile Hibernation media and transported to the laboratory on ice within 1 hr. Patient-derived tumor tissue was cut into small pieces with a scalpel and digested for 30 min at 37°C enzymatically in a mixture consisting of Papain (20 µ/mL, #LK003176, Worthington) and DNase (2000 µ/mL, #LK003170, Worthington). Ovomucoid inhibitor (10 mg/mL, #LK003182, Worthington) was used to stop the enzymatic activity at room temperature and an erythrocyte lysis was performed for further 20 min at room temperature. Seed the primary GBM cells into a GelTrex or Matrigel-coated plastic flask and cultivate in RPMI 1640 (R8758, Sigma) supplemented with 10% FCS (F7524, Sigma) and 1% penicillin/streptomycin (P0781, Sigma). Two primary cell lines (T76, H34) isolated from patient tumor tissue were included in this experimental study. This study was approved by the Research Ethics Committee of The Second Affiliated Hospital of Anhui Medical University. Informed consent was obtained from all the patients.
Lk003170
Manufactured by Worthington
Sourced in United States
The LK003170 is a laboratory equipment product. It serves as a core function for laboratory operations, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Lk003176, by Worthington (3 mentions)
Am2696, by Thermo Fisher Scientific (2 mentions)
Pi 28324, by Thermo Fisher Scientific (2 mentions)
Tcl buffer, by Qiagen (2 mentions)
Facsaria, by BD (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
P0781, by Merck Group (1 mentions)
Lk003182, by Worthington (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Dnase, by Worthington (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Il 34, by Miltenyi Biotec (1 mentions)
Dnase 1, by Worthington (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
5 protocols using lk003170
1
Isolation and Culture of Primary GBM Cells
2
Microglia Isolation from Human Brain
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Fresh postmortem adult human brain tissue was provided by the Netherlands Brain Bank (NBB). All subjects gave their informed consent for inclusion before they participated in the study. Primary microglia were isolated according to the protocol described before with some minor modifications for human brain tissue [49 (link)]. Organoids were dissociated into a single-cell suspension by enzymatic dissociation using papain (18.6 U/mL, Worthington, LK003176, Columbus, OH, USA) and DNAse 1 (337 U/mL, Worthington, LK003170) according to the protocol published before [45 (link)].
Microglia enrichment was achieved by positive selection for CD11b expression, using magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Primary microglia (pMG) or organoid-derived microglia (oMG) were cultured in poly-L-lysine hydrobromide (PLL)-coated 96-well plates (1 × 105 cells/well) in microglia medium (RPMI 1640; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% FCS, 1% penicillin-streptomycin (Gibco Life Technologies, USA) and 100 ng/mL IL-34 (Miltenyi Biotec, Germany)).
Microglia enrichment was achieved by positive selection for CD11b expression, using magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Primary microglia (pMG) or organoid-derived microglia (oMG) were cultured in poly-L-lysine hydrobromide (PLL)-coated 96-well plates (1 × 105 cells/well) in microglia medium (RPMI 1640; Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% FCS, 1% penicillin-streptomycin (Gibco Life Technologies, USA) and 100 ng/mL IL-34 (Miltenyi Biotec, Germany)).
3
Single-cell Transcriptome Profiling with Fluidigm and Smart-seq2
4
Single-cell Transcriptome Profiling with Fluidigm and Smart-seq2
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Cells were dissociated using accutase supplemented with DNase (Worthington Biochemical, LK003170). A 100-μm nozzle was used for sorting. Cells were sorted by BD Facs Aria, 20 psi, 40–100 ev/s into 96-well plates (one cell per well) containing 5 μL lysis buffer. For Fluidigm Biomark, cells were single-cell sorted into 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final, Ambion, AM2696), and 0.5% NP40 (Thermo Scientific, PI-28324). For Smart-seq2 library prep, cells were single-cell sorted into 5 μL 1×TCL buffer (QIAGEN, 1031576). After sorting, plates were immediately sealed, spun down for 5 min at 550 × g, and flash frozen on dry ice. Sorted cells were then stored at −80°C. Details on single-cell expression profiling and transcriptomics analysis are provided in the Supplemental Experimental Procedures . To compare the piN cells to publicly available single-cell reference datasets (Pollen et al., 2014 (link), Darmanis et al., 2015 (link)), we performed integrated analysis using Seurat version (v.)2.1
5
Dissociation of Organoid Cultures
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Five organoids per 60 mm dish were washed three times with DPBS (ThermoFisher, 14190094) and immersed in 6 mL of papain (18.6 U/mL, Worthington, LK003176) and DNAse 1 (337 U/mL Worthington, LK003170) in DMEM/F12. Mechanical dissociation using scalpels was followed by a 30 min incubation at 37 °C on a shaker. In all, 2% FBS was added to stop the enzymatic reaction and the resulting single-cell suspension was centrifuged followed by an additional incubation with DNAse 1 (337 U/mL, Roche Diagnostics, 11284932001) in MACs buffer (PBS pH 7.4 (Gibco Life technologies, MA)), 2 mM EDTA (Sigma-Aldrich, The Netherlands) and 1% FBS) at room temperature for 15 min.
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