Sep 14, 2025

Public workspaceFlow Cytometry Protocol for Cell Death Analysis in Glioblastoma Organoids: A Technical Note

Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.q26g79op8vwz/v1
  • Anna-Laura Potthoff1,2,3,
  • Meng-Chun Hsieh1,2,
  • Ahmad Melhem1,2,
  • Susanna S. Ng4,
  • Barbara E. F. Pregler1,2,
  • Annika Vieregge1,2,
  • Markus Raspe1,2,
  • Lea L. Friker2,3,4,
  • Thomas Zeyen2,5,
  • Julian P. Layer2,4,6,
  • Andreas Dolf7,
  • Marieta I. Toma8,
  • Andreas Waha3,
  • Torsten Pietsch3,
  • Mike-Andrew Westhoff9,
  • Hartmut Vatter1,
  • Michael Hölzel4,
  • Ulrich Herrlinger2,5,
  • Matthias Schneider1,2
  • 1Department of Neurosurgery, University Hospital Bonn, Bonn, Germany;
  • 2Brain Tumor Translational Research Group, University Hospital Bonn, Bonn, Germany;
  • 3Institute of Neuropathology, University Hospital Bonn, Bonn, Germany;
  • 4Institute of Experimental Oncology, University Hospital Bonn, Bonn, Germany;
  • 5Department of Neurooncology, Center for Neurology, University Hospital Bonn, Bonn, Germany;
  • 6Department of Radiation Oncology, University Hospital Bonn, Bonn, Germany;
  • 7Flow Cytometry Core Facility, Medical Faculty, University of Bonn, Bonn, Germany;
  • 8Department of Pathology, University Hospital Bonn, Bonn, Germany;
  • 9Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Ulm, Germany
Anna-Laura Potthoff
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g79op8vwz/v1
External link: https://doi.org/10.1371/journal.pone.0327660
Protocol Citation: Anna-Laura Potthoff, Meng-Chun Hsieh, Ahmad Melhem, Susanna S. Ng, Barbara E. F. Pregler, Annika Vieregge, Markus Raspe, Lea L. Friker, Thomas Zeyen, Julian P. Layer, Andreas Dolf, Marieta I. Toma, Andreas Waha, Torsten Pietsch, Mike-Andrew Westhoff, Hartmut Vatter, Michael Hölzel, Ulrich Herrlinger, Matthias Schneider 2025. Flow Cytometry Protocol for Cell Death Analysis in Glioblastoma Organoids: A Technical Note. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79op8vwz/v1
Manuscript citation:
Potthoff A, Hsieh M, Melhem A, Ng SS, Pregler BEF, Vieregge A, Raspe M, Friker LL, Zeyen T, Layer JP, Dolf A, Toma MI, Waha A, Pietsch T, Westhoff M, Vatter H, Hölzel M, Herrlinger U, Schneider M (2025) Flow cytometry protocol for cell death analysis in glioblastoma organoids: A technical note. PLOS One 20(9). doi: 10.1371/journal.pone.0327660
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 10, 2025
Last Modified: September 14, 2025
Protocol Integer ID: 220179
Keywords: Flow cytometry protocol, Cell death analysis, Glioblastoma organoids, flow cytometry protocol for cell death analysis, cell death in glioblastoma organoid, flow cytometry protocol, using flow cytometry, flow cytometry, glioblastoma organoid, throughput applications in translational cancer research, cell death analysis, flow cytometric analysis, cytometric analysis, translational cancer research, cell death rate, extent of cell death, cell death, dna fragmentation, cell suspension
Abstract
Here, we present a protocol to quantify cell death in glioblastoma organoids (GBOs) using flow cytometry. Single-cell suspensions are generated through combined enzymatic and mechanical dissociation, followed by permeabilization and nuclear staining with propidium iodide or Hoechst 33258 to detect DNA fragmentation.
Following treatment with temozolomide (TMZ) and lomustine (CCNU) for up to 288 hours, flow cytometric analysis revealed cell death rates of up to 63%, with CCNU demonstrating greater efficacy than TMZ. The extent of cell death varied between GBO populations but was consistent across replicates derived from the same GBO population. The protocol is scalable, cost-effective, and suitable for high-throughput applications in translational cancer research.
Guidelines
Protocol for flow cytometric analysis

Here, we provide a protocol for measuring cell death in GBOs after treatment with two different chemotherapeutic drugs. For us, measurement time points 144 hours and 288 hours after treatment initiation are sufficient. However, depending on desired drugs, earlier measurement time points might be more suitable. The protocol can be adapted for organoids from other models or tumor entities. Therefore, parameters such as the number of organoids needed, incubation times, and other steps need to be adjusted. We recommend processing no more than 12 samples at a time to ensure consistency and manageability. Within the combined enzymatic and mechanical dissociation step, it is important to resuspend the suspension and observe it under the microscope every few minutes to monitor the progress of the dissociation process.
Materials
Table of materials

ABCD
Reagent name Manufacture Category number Note
GBO cultured medium
DMEM/F12 Thermo Fisher Scientific 11320033
Neurobasal medium Thermo Fisher Scientific 21103049
MEM-NEAAs solution (100x) Thermo Fisher Scientific 11140050
GlutaMAX supplement (100x) Thermo Fisher Scientific 35050061
N2 supplement (100x) Thermo Fisher Scientific 17502048
B27 supplement (50x), minus vitamin A Thermo Fisher Scientific 12587010
Penicillin-streptomycin (100x) Thermo Fisher Scientific 15070063
Human insulin solution Sigma-Aldrich I9278
2-Mercaptoethanol Thermo Fisher Scientific 219850231:1000 dilution in GBO medium

  • ReagentDMEM/F12Thermo Fisher ScientificCatalog #11320033
  • ReagentNeurobasal™ MediumGibco - Thermo Fisher ScientificCatalog #21103049
  • ReagentMEM Non-Essential Amino AcidsGibco - Thermo Fisher ScientificCatalog #11140050
  • ReagentGlutaMAX™ SupplementGibco - Thermo Fisher ScientificCatalog #35050061
  • ReagentN-2 Supplement (100X)Thermo FisherCatalog #17502048
  • ReagentB-27™ Supplement (50X) minus vitamin AThermo Fisher ScientificCatalog #12587010
  • ReagentPenicillin-Streptomycin (5,000 U/mL)Thermo FisherCatalog #15070063
  • ReagentInsulin solution humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9278
  • Reagent2-MercaptoethanolGibco - Thermo Fisher ScientificCatalog #21985023

ABCD
PI buffer
Propidium iodide (PI) stock (1mg/1ml) SIGMA P4170 1mg/ml stock in Ampuwa water dilution
Triton X-100 SIGMA T8787 0.10%
Trisodium citrate Merck KGaA 1.37042.1000 0.10%
  • ReagentPropidium Iodide (PI) Merck MilliporeSigma (Sigma-Aldrich)Catalog #P4170
  • ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787

ABCD
Reagent nameManufactureCategory number Note
Trypsin-EDTA (0.5%) gibco15400054no phenol red
CytoTox 96® Non-Radioactive Cytotoxicity Assay PromegaG1780
Hoechst 33258SIGMAB1155_25MG10 mg/ml stock in Ampuwa water dilution

  • ReagentTrypsin-EDTA (0.5%) no phenol redThermo Fisher ScientificCatalog #15400054
  • ReagentCytoTox 96(R) Non-Radio. Cytotoxicity Assay, 10X96 well pltPromegaCatalog #G1780
ABCD
Drug nameManufactureCategory number Note
Temozolomide (TMZ)TOCRIS 2706
Lomustine (CCNU) Merck L5918
ABCD
Equipment nameManufactureCategory number Note
6 well-plate (suspension) Sarstedt 83.3920.500
1000ul filtered tip Sarstedt70.3060.255
Eppendorf® Combitips Advanced® 5ml Eppendorf 30089456
15ml falconThermo Fisher Scientific 339650
50ml falconThermo Fisher Scientific 339652
Cell Strainer 70µm Corning7201431
Flow Cytometry Tube 5 ml Sarstedt 55.157975 x 12 mm, PS
Orbital shakerThermo Fisher Scientific
Magnetic stirrer
FACS Canto llBecton Dickinson, Heidelberg, Germany
Centrifuge 5810 R Eppendorf
µQuant microplate spectrophotometer Biotek Instruments, USA
ABCD
Software nameManufactureCategory number Note
FlowJoFlowJo LLC, Ashland, OR, USA V10.4
GraphPad PRISM GraphPad Software, Boston, M, USA V9.5.1
Adobe Illustrator 2023 Adobe Inc., Dublin Republic of IrelandV27.7



Troubleshooting
Treatment (Day 0)
Preparation of GBO Culture Medium
Prepare the GBO culture medium as previously described by Jacob et al. [1]. The medium consists of a 1:1 mixture of DMEM:F12 medium (Amount235 mL ) and Neurobasal medium (Amount235 mL ), supplemented with the following components:

  • Amount5 mL of MEM-NEAAs solution (100×),
  • Amount5 mL of GlutaMAX (100×),
  • Amount5 mL of penicillin-streptomycin (100×),
  • Amount5 mL of N2 supplement (100×),
  • Amount10 mL of B27 minus vitamin A (50×),
  • Amount125 µL of human recombinant insulin.

Pipetting
Sterilize the medium by filtration through a 0.2-μm PES membrane. Immediately before use, add 2-mercaptoethanol at a 1:1000 dilution. Refer to the material table for additional details.

Note
  • The complete GBO medium (without 2-mercaptoethanol) can be stored at Temperature4 °C for up to 4 weeks.
  • 2-mercaptoethanol must be added to the GBO medium just before use.

Temperature
GBO Selection and Treatment Setup
GBO Selection:

  • Select 8-10 GBOs per treatment condition based on size (optimal size range: 500–700 µm).
  • Ensure all selected GBOs are of approximately similar size to avoid inconsistencies in drug penetration and inaccurate results.
  • Measure the diameter of all GBOs and calculate the mean diameter per treatment condition to ensure there is no significant difference between groups. If the organoids are not perfectly round, record the longest diameter.
Plate Setup:

  • Transfer the selected 8-10 GBOs to individual wells of a non-coated 6-well plate, with each treatment condition assigned to a separate well.
  • Perform at least technical duplicates (i.e., two wells per condition) to ensure reproducibility.

Note
  • For better control and safety, prepare extra wells for flow cytometry acquisition if performing the procedure for the first time or using new GBOs populations (new patient material).
  • Always include an untreated control group to assess the level of spontaneous cell death (usually higher in GBOs than in 2D cell culture populations).

Medium Addition:

  • Add Amount4 mL of GBO culture medium to the control wells.
  • Add Amount4 mL of GBO medium containing the desired drug concentration to the treatment wells (TMZ/CCNU 100µM concentration: 1:1000 from a Concentration100 millimolar (mM) stock (Amount4 µL for Amount4 mL )).

Pipetting
Incubation:

Place the 6-well plate on an orbital shaker set to Shaker120 rpm inside a humidified incubator (Temperature37 °C , 5% CO2, 90% relative humidity).

Incubation
Mix
Temperature
Medium Change (Every 48 hours)
2m
The medium must be changed every 48 hours due to the high cell density within the GBOs and the rapid proliferation rate of individual cells.
Removal of Old Medium:

  • Remove the 6-well plate from the incubator.
  • Allow the organoids to settle by tilting the plate at a slight angle for 1–Duration00:02:00 .
  • Gently aspirate the old medium from each well using a 1000 µl filtered tip and transfer it into a 15 ml centrifuge tube.
  • Store the centrifuge tube containing the collected medium at Temperature-20 °C (collected medium will be used later for flow cytometry and optionally for the LDH assay).

Note
  • Not including the old medium in flow cytometry measurement may lead to underestimation of cell death, as dead cells may detach and be in suspension.
  • GBOs are sticky and may adhere to the pipette tip during medium change. If any GBO adhered to the tip, reload it back into the well and re-do the procedure.

2m
Pipetting
Temperature
Addition of Fresh Medium:

  • Add Amount4 mL of fresh drug-containing medium to each well, as described in Step 2.3.

Note
  • Repeat the medium change every 48 hours until the designated measurement time point is reached.
  • Pool the collected medium from each condition into the corresponding centrifuge tube and store it at Temperature-20 °C .

Pipetting
Temperature
GBO Dissociation for Subsequent PI Staining and Cell Death Measurement at the Designated Time Point
25m
Preparation of propidium iodide (PI) Buffer:

  • Combine Amount5 mL of PI stock (1 mg/ml) with Amount95 mL of 0.1% sodium citrate to achieve a final concentration of 50 mg/l.
  • Add Amount100 µL of Triton X-100 (0.1%) and mix thoroughly on a magnetic stirrer until complete dissolution is achieved.

Note
  • You may prepare the buffer one day in advance.
  • Cover the PI buffer container with aluminum foil to protect it from light.
  • Store at Temperature4 °C in darkness. The buffer can be stored for up to 6 months.
  • Always wear gloves when handling PI buffer, as it is mutagenic.

Additional Preparations:

  • Pre-warm trypsin in a water bath to Temperature37 °C .
  • Prepare an ice bucket.

Pipetting
Temperature
Thawing Collected Medium:

On the day of measurement, thaw the centrifuge tubes containing the collected medium by placing them at TemperatureRoom temperature .

Note
Start thawing 1–2 hrs in advance, and place the centrifuge tubes TemperatureOn ice once thawed.

Centrifigation
Temperature
Collection of Final Medium:

Collect the last medium from the wells and transfer it to the corresponding thawed centrifuge tubes.

Note
Remove as much medium as possible from each well. If necessary, use a smaller tip to remove the remaining medium without aspirating GBOs.

Washing GBOs:

Wash the GBOs with Amount2 mL of DPBS to remove any residual medium. Gently tilt the plate back and forth and side to side a few times. Discard the DPBS and repeat the wash step again.

Pipetting
Wash
Enzymatic Dissociation:

  • Add Amount2 mL of warm trypsin (0.05%) to each well for enzymatic dissociation. Tilt the plate or gently swirl to ensure that the GBOs are completely immersed.
  • Place the plate on the orbital shaker in the incubator at Temperature37 °C for Duration00:10:00 .

10m
Incubation
Pipetting
Temperature
Centrifugation of Collected Medium:

  • While waiting, centrifuge all thawed centrifuge tubes at Centrifigation4000 rpm, 4°C, 00:10:00 .
  • Discard the supernatant until 3 ml remains in each centrifuge tube. Keep the tubes TemperatureOn ice .

10m
Centrifigation
Temperature
Mechanical Dissociation:

  • After 10 minutes, speed up the dissociation process by gently pipetting up and down 5–10 times using a wide-bored 1000 µl pipette tip.
  • Repeat mechanical dissociation every 5-10 minutes, returning the plate to the incubator between steps.

Note
  • Enzymatic dissociation alone is insufficient; combine it with gentle mechanical dissociation to effectively and rapidly reach single-cell suspension.
  • Avoid vigorous pipetting to prevent cell damage or death.

Pipetting
Monitoring Dissociation:

Monitor dissociation progress under a microscope after each resuspension step until no cell clumps are visible.

Note
  • Dissociation time varies with GBO size and density, and between different GBO populations (different patient material).
  • Avoid over-trypsinization; for tested GBOs, the mean dissociation time was ~Duration00:30:00 (range: 20–45 minutes).

Filtration:

  • Filter the cell suspension through a 70 µm strainer to avoid small cell aggregates.

Neutralization of Trypsin:

  • Transfer the filtered single-cell suspension to the corresponding centrifuge tube containing 3 ml of supernatant (from Step 4.5) to slow the trypsin reaction.
Addition of HBSS (optional):

  • Add Amount2 mL of HBSS to the centrifuge tubes to maintain osmolality and pH.

Pipetting
Centrifugation:

  • Centrifuge at Centrifigation1300 rpm, 4°C, 00:05:00 .

5m
Centrifigation
Temperature
Discarding Supernatant:

  • Gently discard the supernatant by pouring. Some residual medium is acceptable.
Resuspension:

  • Resuspend the cell pellet in the remaining volume (typically 300–350 µl) in the 15 ml centrifuge tube.
Staining of Single Cell Suspension with PI-Buffer
1h
Transfer Single Cell Suspension:

  • Transfer the entire single-cell suspension into a FACS tube (round-bottom FACS tube for BD FACS Canto™ II, BD Biosciences).
Add HBSS:

  • Add Amount100 µL HBSS to prevent aggregation and maintain osmolality.
Pipetting
Divide Cell Suspension:

  • Divide the single-cell suspension equally into two FACS tubes (approx.Amount150 µL per tube), one for PI staining and the other one for Hoechst staining (see Step 11).
Pipetting
Add PI-Buffer:

  • Add Amount100 µL of the PI-buffer to each tube.
Pipetting
Incubation:

  • Incubate the tubes TemperatureOn ice for 20-Duration00:30:00 .

Note
  • Perform flow cytometric measurement within 4 hours after adding PI-buffer. For best results, start measurement immediately after incubation and complete within 1 hour to prevent cell aggregation.
  • Keep all samples TemperatureOn ice until measurement.

30m
Incubation
Data acquisition:
Set up the flow cytometry device (e.g., BD FACSCanto™ II, Becton Dickinson) for analysis.
Suggested settings (may vary between organoid model/GBO population and device): FCS threshold: 800; Voltages: SSC: 243, FCS: 132, PI: 291.

Note
  • Set an accurate threshold to exclude debris while ensuring the sub-G1 peak (containing dead cell information) is not cut off.
  • Check for doublets while acquisition (e.g., plot FSC-A vs. FSC-H).

Staining of Single Cell Suspension with Hoechst-Buffer
30m
Preparation of Hoechst Buffer

  • Dilute Hoechst 33258 in HBSS to achieve a 1:5000 dilution ratio.
Add Hoechst Buffer:

  • Add the same volume of Hoechst buffer to the single-cell suspension to achieve a final dilution of 1:10000 (Amount150 µL in this protocol).

Pipetting
Incubation:

  • Incubate TemperatureOn ice for 15–Duration00:30:00 .

30m
Incubation
Temperature
Data acquisition:

  • Settings vary between organoid model/GBO population and device (example settings for our GBO populations and BD FACS Canto II, BD Biosciences: FCS threshold: 800; Voltages: SSC: 243, FCS: 132, 450/50 nm violet: 269.

Note
  • Use the same settings for threshold as well as SSC and FSC voltages as applied during the PI measurement.

Protocol references
1. Jacob F, Ming GL, Song H. Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing. Nature protocols. 2020;15(12):4000-33. Epub 20201109. doi: 10.1038/s41596-020-0402-9. PubMed PMID: 33169003.