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JoVE Journal Cancer Research

Cancer Research

Revealing the Ferroptotic Phenotype of Medulloblastoma

Published: March 15, 2024 doi: 10.3791/66645
1, 1, 1, 1,2, 1, 1
1Medical Biology department, Centre Scientifique de Monaco (CSM), 2Institute for Research on Cancer & Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, University Côte d’Azur

Abstract

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named "ferroptosis." The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic "bubbling" phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Introduction

Ferroptosis is a newly contextualized, reactive oxygen species (ROS)-dependent type of cell death1. Besides ROS, iron plays a crucial role(s) in this type of cell death, hence the name2. The final and executive step of ferroptosis is the iron-catalyzed accumulation of oxidative damage of lipids in the plasma membrane that eventually leads to compromised membrane integrity and selective permeability, and, finally, cell death by bubbling. Lipid hydroperoxidation event is a naturally occurring phenomenon; however, its propagation throughout the cellular membrane is prevented by the antioxidant defense of the cell. The major player in this context is Se-protein glutathione peroxidase 4 (GPx4), which can approach the membrane and convert lipid hydroperoxides into their less toxic alcohol derivatives3. The reducing power for GPx4 is mainly, but not exclusively, provided by glutathione (GSH), a tripeptide composed of the non-essential amino acids: glycine, glutamate, and cysteine. The rate-limiting amino acid for the biosynthesis of GSH is cysteine4. Although cysteine is classified as a non-essential amino acid, its requirements can easily exceed its internal production in highly proliferative cells (such as cancer cells). It thus has been re-classified in the group of semi-essential amino acids. The necessary import of cysteine occurs mainly through the Xc- system, which allows the import of the oxidized (dominant) form of cysteine (aka cystine) at the expense of glutamate export5. The Xc- system is composed of a Na+-independent, Cl--dependent transport subunit, known as xCT, and a chaperon subunit, known as CD98. Until recently, the anti-ferroptotic properties of the xCT-GSH-GPx4 axis have been seen as unique and irreplicable6. However, in 2019, an alternative anti-ferroptotic pathway, consisting of ubiquinol (Coenzyme Q10) and its regenerative enzyme - ferroptosis suppressor protein 1 (FSP1), has been described7,8. Soon afterward, yet another lipid hydroperoxide detoxifying system involving GTP cyclohydrolase-1/tetrahydrobiopterin (GCH1/BH4) has been reported9. Nonetheless, the central role of the xCT-GSH-GPx4 axis in the prevention of ferroptosis seems not to be challenged.

Over the past decade, ferroptosis has been extensively studied in a variety of tumor types, showing great potential as an anti-cancer strategy (reviewed by Lei et al.10). Furthermore, it has been reported that cancer cells exhibiting high resistance to conventional chemotherapeutics and/or a propensity to metastasize are surprisingly sensitive to ferroptosis inducers, such as inhibitors of GPx411,12,13. However, in the context of brain tumors, the potential of ferroptotic inducers remains largely understudied. While this type of cell death has been closely associated with cerebral ischemia-reperfusion injury14and neurodegenerative diseases15, its potential in the context of brain tumors has mainly been limited to glioblastoma, the most common malignant craniocerebral tumor (reviewed by Zhuo et al.16). On the other hand, the sensitivity of medulloblastoma, the most common malignant pediatric brain tumor and a leading cause of childhood mortality, to ferroptosis inducers remains largely unexplored. To the best of our knowledge, there is scarce peer-reviewed literature linking ferroptosis and medulloblastoma. Nonetheless, some studies have revealed that iron plays a crucial role in the survival, proliferation, and tumorigenic potential of both medulloblastoma and glioblastoma cancer stem cells (CSCs)17,18, potentially rendering them more vulnerable to ferroptosis induction. This is particularly significant as medulloblastoma is notorious for its subpopulation of CSCs, or tumor-initiating/propagating cells, which appear to be largely responsible for tumor chemoresistance, dissemination, and relapse19.

Sensitivity to ferroptosis induction is typically investigated by measuring lipid hydroperoxide content/accumulation, which may or may not lead to cell death. The most commonly used ferroptosis inducers are (1) erastin, an inhibitor of the xCT transporter20,(2) RSL3, an inhibitor of the GPx4 enzyme2, and/or (3) iron donors, such as ferro-ammonium citrate (FAC)21. Lipid hydroperoxide content is assessed using the selective probe BODIPY 581/591 C1122, which has excitation and emission maxima at 581/591 nm in its reduced state. Upon interaction with and oxidation by lipid hydroperoxides, the probe shifts its excitation and emission maxima to 488/510 nm. Typically, a significant increase in lipid hydroperoxide content precedes ferroptotic cell death. Since ferroptosis is not a programmed cell death, there is no molecular signaling cascade leading to its execution. Therefore, the only way to confirm it is to monitor lipid hydroperoxide content and use specific inhibitors for this type of cell death, such as ferrostatin 123. Ferrostatin 1 is a lipophilic antioxidant that can penetrate the lipid compartment of the cell and detoxify lipid hydroperoxides, thereby preventing ferroptotic events.

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Protocol

The present study was conducted using DAOY wild-type (WT) medulloblastoma cell lines, which were cultured at 37 °C with 5% CO2 in DMEM medium supplemented with 8% FBS. The xCT-deleted cell line was maintained under the same conditions, with experiments carried out in media supplemented with 1 mM N-acetylcysteine (NAC). The cells were regularly screened for mycoplasma using a commercially available Mycoplasma Detection Kit (see Table of Materials) and were cultured up to the 10th passage.

1. Harvesting and seeding the cells

NOTE: All steps are performed using sterile aseptic techniques in a tissue culture laminar flow hood. DAOY medulloblastoma cells are adherent, meaning all unattached cells can be discarded by washing with phosphate-buffered saline (PBS) without Ca2+ and Mg2+.

  1. Take the stock dish (Petri dish, 100 mm diameter) with the cells and remove the floating cells and medium from the plate using an aspirator.
  2. Add 5-10 mL of PBS to the dish, wash the bottom with circular movements of the dish, and then remove the PBS from the plate using an aspirator.
  3. Add 1 mL of trypsin (10x dilution) to the dish. Incubate the plate until gentle tapping of the plate dislodges the cells. Take 10 mL of DMEM culture medium supplemented with 8% FBS and mechanically harvest the cells off the dish.
  4. Transfer the cell suspension into a 15 mL tube.
  5. Take 500 µL of the cell suspension and put it in a cuvette for counting. Count the number of cells per mL of cell suspension. An automated cell counter was used for this study (see Table of Materials).
  6. Calculate the volume necessary to take from the cell suspension to have 1,00,000 cells.
  7. Take a 6-well plate and add the calculated volume of the cell suspension into each well, then add DMEM culture media supplemented with 8% FBS to 2 mL in each well.

2. Treatment of the cells

NOTE: The control and treatments are conducted in triplicate. The groups are as follows: Control (DMSO), 1 µM of erastin, 0.3 µM of RSL3, 250 µM of FAC, 2 µM of Ferrostatin 1, 1 µM of erastin + 2 µM of Ferrostatin 1, 0.3 of µM RSL3 + 2 µM of Ferrostatin 1, 250 µM of FAC + 2 µM of Ferrostatin 1. Four 6-well plates are needed for the experiment, as indicated in Table 1). The commercial details of all the necessary reagents are listed in the Table of Materials.

  1. 24 h post-seeding, add the corresponding treatment to the well with the cells.
  2. Leave the dishes at 37 °C and 5% CO2 in the incubator.

3. Lipid hydroperoxides staining of the treated cells with BODIPY 581/591 C11 probe

NOTE: The stock solution of the lipid hydroperoxide-specific probe is prepared in DMSO at a concentration of 1 mM. Aliquots of the stock solution are stored at -20 °C in non-transparent tubes. For staining, prepare a 2 µM working solution of the probe in DMEM media supplemented with 8% FBS.

  1. 6 h post-treatment, observe the cells under a light microscope (cell rounding should be visible).
  2. Remove the dishes from the incubator and place them in a tissue culture laminar flow hood.
    Using an aspirator, remove the media and floating (dead) cells.
  3. Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.
  4. Add 2 mL of the working solution of the probe (2 µM final concentration in DMEM supplemented with FBS).
  5. Leave the dish in the incubator at 37 °C and 5% CO2 for 30 min in the dark (the plates can be wrapped in aluminum foil).

4. Flow cytometry analysis of lipid hydroperoxide content in the treated cells

NOTE: All the following steps are performed in the dark (no lights in the laminar flow hood).

  1. Take the dishes out of the incubator and place them in a tissue culture laminar flow hood.
    Using an aspirator, remove the staining solution.
  2. Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.
  3. Repeat the washing step one more time and aspirate any remaining PBS from the well using an aspirator.
  4. Add 150 µL of a commercially available cell dissociation reagent (see Table of Materials) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Take 250 µL of FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and mechanically harvest the cells off the wells.
  5. Transfer the cells into the corresponding (previously marked) FACS tube with the filter cap. Place the tube with the cells on ice in the dark until the analysis is performed (analysis should be conducted within an hour after staining).
    NOTE: FACS machine setup and calibration depend on the machine used (see Table of Materials).
  6. Create a new experiment and give it a name (Ferroptosis in DAOY - lipid hydroperoxides).
  7. In the experiment design, choose the laser (488 nm) and the filter (FITC).
  8. In view data, select the proper cell size (>12 µm), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.
  9. Add a new plot - histogram, with FITC-A on the y-axis and logarithmic scale.
  10. Vortex the samples in the FACS tube, place them in the FACS machine and load the sample. Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.

5. Propidium iodide (PI) staining of the dead cells upon treatment

NOTE: The experiment design is exactly the same as for lipid hydroperoxide measurement (see step 1 and step 2).

  1. 24 h post-seeding, take the dishes out of the incubator and place them in the laminar flow hood.
  2. Collect the media (with dead cells) in a 15 mL tube.
  3. Add 1 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then collect the PBS into the tube with the respective media.
  4. Add 200 µL of trypsin (10x dilution) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Use the respective media + PBS to harvest the cells off the wells.
  5. Centrifuge the cell suspension at 180 x g for 10 min at room temperature. Remove the supernatant using an aspirator.
  6. Resuspend the cell pellet in 300 µL of FACS buffer and place it on ice until analysis.
  7. Just before the analysis, add PI solution (see Table of Materials) to a final concentration of 2 µg/mL.

6. Flow cytometry analysis of dead cells 24h post-treatment

NOTE: FACS machine settings and calibration are done as previously indicated (see step 4).

  1. Create a new experiment and give it a name (Ferroptosis in DAOY - cell death).
  2. In the experiment design, choose the laser (488 nm) and the filter (PE).
  3. In view data, select the proper cell size (>12 µm), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.
  4. Add a new plot - histogram, with PE-A on the y-axis and logarithmic scale.
  5. Vortex the samples in the FACS tube, place them in the FACS machine and load the sample.
  6. Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.

7. Flow cytometry analysis of lipid hydroperoxide content in the DAOY xCT-/- cells

NOTE: xCT-/- cells have been generated as previously described24.

  1. For this experiment, seed the cells as indicated in step 2, but instead of the treatment, supplement the media with 1 mM NAC overnight.
  2. The next day, maintain NAC in one group and remove it from the other.
  3. Perform the flow cytometry analysis of lipid hydroperoxide content in the exact same way as described in step 6.

8. Analysis of the flow cytometer results

  1. Open the .fcs document in the FlowJo software (see Table of Materials).
  2. Double click on the individual file to open all events (not only FCS singlets) recorded in the individual sample (SSC-A/FSC-A dot plot). Since the settings are such that the gating done on the machine is not preserved, it is necessary to make the gate once again and name the population (e.g., DAOY WT).
  3. Double click on the gated population to open another window with the histogram.
  4. Change the Y-axis to the fluorescent filter used (Comp-FITC/PE-A).
  5. Change the X-axis to modal (normalize each peak to its mode, i.e., to % of the maximum number of cells found in a particular bin).
  6. Copy the histogram into the layout editor. Repeat this for all the samples, and every time a new histogram is pasted in the layout editor, drag it over the previous one so that the histograms are overlaid (half-offset).

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Representative Results

The medulloblastoma cell line DAOY was cultured in a standard DMEM medium supplemented with 8% FBS until it reached approximately 60% confluency. On the day of the experiment, cells were harvested, and 1,00,000 cells per well were plated in 6-well plates, according to Table 1. The following day, cells (in triplicate) were treated with either 1 µM of erastin, 0.3 µM of RSL3, or 250 µM of FAC. The plates were then placed in the incubator at 37 °C and 5% CO2. After 6 h, cells were observed under the microscope to detect bubbling cells, as indicated by the arrows in Figure 1. This served as an indicator that the drug was effective in inducing ferroptosis before the cells were prepared for lipid hydroperoxide staining and subsequent FACS analysis.

For the detection of lipid hydroperoxide content, the specific dye BODIPY 581/591 C11 was used at a final concentration of 2 µM for 30 min. As this dye is redox-sensitive, it was necessary to remove any treatment from the cells and wash them with pre-warmed PBS. After half an hour of staining, cells were washed twice with PBS, detached using the cell detachment solution, and prepared for FACS analysis. Data obtained at this step showed increased green fluorescence of the dye in the samples treated with all three drugs (Figure 2). However, the most potent effect was observed with 0.3 µM of RSL3 (Figure 2B), while 1 µM of erastin (Figure 2A) and 250 µM of FAC (Figure 2C) showed a somewhat lesser effect after 6 h of treatment.

To establish that the detected lipid hydroperoxide accumulation leads to cell death, specifically ferroptosis, the same treatments outlined in Table 1 were applied for 24 h. For the analysis of dead cells by flow cytometry, both the cells and supernatant were harvested. The live and dead cells were pelleted by centrifugation and then resuspended in FACS buffer for subsequent flow cytometry analysis. The data presented in Figure 3 demonstrated that all three treatments induced massive cell death, which was completely prevented by using the ferroptosis-specific inhibitor, ferrostatin-1. This clearly supports the occurrence of ferroptotic cell death upon xCT inhibition, GPx4 inhibition, or iron overload in medulloblastoma cells.

Given that the three drugs showed somewhat different effects on lipid hydroperoxide accumulation, likely due to differences in potency, a genetic approach was employed to investigate the full potency of the ferroptotic inducers. Previously obtained DAOY xCT-/- cells (Supplementary Figure 1A) were cultured in the same media as their WT counterparts but with NAC supplementation. This positive selection allowed the xCT-/- cells to grow in culture. However, 6 h after NAC was removed from the culturing media, cells began to accumulate lipid hydroperoxides to the same level as WT cells treated with RSL3 (Figure 4). Similarly to pharmacological inhibition, the removal of NAC from the media induced the characteristic bubbling of xCT-/- cells after 6 h (Supplementary Figure 1B).

Figure 1
Figure 1: Ferroptotic phenotype of medulloblastoma cells. Representative micrographs of DAOY WT cells treated (or not) for 6 h with 1 µM of erastin (ERA), 0.3 µM of RSL3 or 250 µM of ferroammonium citrate (FAC) in the presence or not of 2 μM ferroptosis inhibitor Ferrostatin-1 (Ferro-1). White arrows indicate round, "bubbling" cells, indicating a ferroptotic phenotype visible under a light microscope. Magnification: 20x, inserts: 40x. Scale bars: 50 µm, insets: 10 µm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Lipid hydroperoxide as the key marker of ferroptosis in medulloblastoma cells. Representative data of the lipid hydroperoxide staining by flow cytometry in the cells treated for 6 h with (A,B) 1 µM of erastin (ERA), (C,D) 0.3 µM of RSL3, or (E,F) 250 µM of ferroammonium citrate (FAC), in the presence or not of 2 µM ferroptosis inhibitor Ferrostatin-1 (Ferro-1). (A,C,E) Dot plots of the recorded events; (B,D,F) Histogram representation of the lipid hydroperoxide content. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Ferroptotic cell death induction by pharmacological approach. Representative flow cytometry dot plots of propidium iodide-stained cells after 6 h of treatment with 1 µM of erastin (ERA), 0.3 µM of RSL3, or 250 µM of ferroammonium citrate (FAC), in the presence or not of 2 µM ferroptosis inhibitor Ferrostatin-1 (Ferro-1). Gating separates live (Q4) and dead (Q3) populations of the cells. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Ferroptosis induction by genetic approach. Representative data of the lipid hydroperoxide staining by flow cytometry in the DAOY WT and xCT-/- cells after 6 h in the presence or not of 2 µM ferroptosis inhibitor Ferrostatin-1 (Ferro-1). On the left are the histogram representation of the lipid hydroperoxide contents. On the right are the dot plots of the recorded events. Please click here to view a larger version of this figure.

Plate I  CTL I CTL II  CTL III
1 µM erastin I 1 µM erastin II 1 µM erastin III
Plate II  0.3 µM RSL3 I 0.3 µM RSL3 II 0.3 µM RSL3 III
250 µM FAC I 250 µM FAC II 250 µM FAC III
Plate III  2 µM Ferro-1 I 2 µM Ferro-1 II 2 µM Ferro-1 III
1 M erastin + 2 M Ferro-1 I 1 M erastin + 2 M Ferro-1 II 1 M erastin + 2 M Ferro-1 III
Plate IV  0.3 M RSL3 + 2 M Ferro-1 I 0.3 M RSL3 + 2 M Ferro-1 II 0.3 M RSL3 + 2 M Ferro-1 III
250 M FAC + 2 M Ferro-1  I 250 M FAC + 2 M Ferro-1 II 250 M FAC + 2 M Ferro-1 III

Table 1: Treatment details of the control and experimental cells.

Supplementary Figure 1: Ferroptotic cell death of the DAOY xCT-/- cells. (A) Western blot analysis of the xCT level in DAOY WT and xCT-/- cells in DMEM media supplemented or not with 2 µM of Ferrostatin-1. (B) Representative micrographs of the DAOY xCT-/- cells in the presence (left) and absence (right) of 1mM N-acetylcysteine for 6 h. White arrows indicate round, "bubbling" cells, indicating a ferroptotic phenotype visible under a light microscope. Magnification: 40x. Scale bars: 10 µm. Please click here to download this File.

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Discussion

The primary hallmark of ferroptotic cell death is the uncontrolled accumulation of lipid hydroperoxides in the plasma membrane. This oxidative damage may occur in an enzymatic or non-enzymatic manner, but in either case, the reaction is iron-dependent/catalyzed, which explains the name of this type of cell death. Lipid hydroperoxidation is often indirectly estimated by measuring the degradation products of lipid hydroperoxidation, such as 4-hydroxy-2,3-trans-nonenal (4-HNE) or malonaldehyde (MDA). These products, generated by the peroxidative decomposition of polyunsaturated fatty acids, can react with various macromolecules, leading to a wide variety of pathologies, such as neurodegenerative disorders, diabetes, and chronic heart failure, among others. Nonetheless, these are the consequences of the peroxidation event, and significant effort has been invested in developing good lipid hydroperoxide-specific probes.

One frequently used probe is BODIPY 581/591 C11, which also has its disadvantages that should be taken into consideration. Specifically, BODIPY 591/581 fluorescence color exhibits two emission peaks, one in its reduced state (590 nm) and one in its oxidized state (510 nm). The ratio between fluorescence at these two wavelengths is of particular importance if precise quantification is the aim. However, the intensity of single-emission fluorescence of BODIPY 591/581 nm has been widely used throughout the literature1,25,26,27,28, especially as the reduced intensity of red emission at 510 nm is frequently masked by the spillover of the fluorescence of the oxidized molecule22,29. This is one of the major reasons why the fluorescence of BODIPY 591/581 is usually represented in a qualitative manner as a function of single (green) fluorescence intensity.

Another important limitation of this probe is its fluorescence emission suppression by antioxidants in a concentration-dependent manner30. Therefore, staining should be carried out in media without antioxidant supplementation. In the present protocol, ferrostatin 1, a powerful scavenger of lipid hydroperoxides, was used; nonetheless, before the probe was added, ferrostatin 1 was removed, and the cells were washed with PBS so that the probe was not exposed to its antioxidant activity.

Regarding ferroptosis induction, many commercially available drugs could be used. Here, erastin, an irreversible inhibitor of the xCT transporter31, was employed. Given that it binds covalently to the transporter, even higher concentrations could be used (up to 100 µM) for a shorter period of time (5 min), followed by a washing step31. To evaluate the exact kinetics of ferroptosis upon complete inhibition of xCT, xCT-/- cells were utilized in the absence of NAC. A massive accumulation of lipid hydroperoxides was detectable after 6 h, and virtually no viable cells were detectable 24 h post-seeding. As for RSL3, a highly potent inducer of ferroptosis in the nanomolar range, it exhibited a kinetic similar to the one observed with xCT-/- cells. Its mode of action is still rather controversial, but it seems that it does not affect the activity but instead induces degradation of the GPx4 enzyme32,33.

An important aspect of ferroptosis induction in vitro is cell confluency. It has long been observed that a confluent cell layer is much more resistant to cysteine starvation, xCT or GPx4 inhibition, GSH depletion, etc.34. The main reason given is that physical contact, i.e., confluency, leads to the activation of the Hippo pathway, which, in turn, downregulates metabolism and internal production of reactive oxygen species (ROS). Furthermore, the shuttle of reduced cysteine could also contribute to resistance to incomplete xCT inhibition35. In the present study, a concentration of 1,00,000 cells per well (6-well plate) was used in order to maintain a low confluency with cells that are rather big and flat. However, with smaller and round-shaped cells, such as HD-MB03, this number could be increased to 1,50,000-2,00,000 cells per well (6-well plate).

The final limitation of the present study is the detection of cell death by the propidium iodide (PI) method. This method is user-friendly for detecting any type of cell death, and ferroptosis could be distinguished from other types of cell death by using ferroptosis-specific inhibitors, such as ferrostatin-1. PI staining is based on the fact that dying cells lose membrane integrity, allowing PI to penetrate the cells, intercalate between the DNA bases, and emit a fluorescent signal. However, it is worth noting that lipid hydroperoxidation induces membrane "leakage," which allows penetration of PI even before cell death occurs. Thus, estimating the kinetics of ferroptosis based on this method alone is challenging.

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Disclosures

We declare no conflict of interest for the study presented herewith.

Acknowledgments

This work was supported by the government of the Principality of Monaco, as well as by 'Le Groupement des Entreprises Monégasques dans la Lutte contre le cancer' (GEMLUC) and Flavien Foundation, which provided the means for BD FACS Melody purchase.

Materials

Name Company Catalog Number Comments
BODIPY 581/591 C11 Thermo Fisher D3861
Cell counter Beckman Coulter Z1
DMEM medium  Gibco 10569010
Erastin Sigma-Aldrich E7781-5MG
Ferroamminium citrate Acros Organics 211842500
Ferrostatin-1 Sigma-Aldrich SML0583-25MG
Fetal bovin serum (FBS) Dominique Dutcher 500105N1N
Flow Cytometer BD Biosciences FACS Melody
Gibco StemPro Accutase Cell Dissociation Reagent Thermo Fisher 11599686
N-acetylcysteine Sigma-Aldrich A7250
PlasmoTest Mycoplasma Detection Kit InvivoGen rep-pt1
propidium iodide Invitrogen P3566
RSL3 Sigma-Aldrich SML2234-25MG
Trypsin - EDTA 10X - 100 mL Dominique Dutcher X0930-100

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References

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Segui, F., Daher, B., Gotorbe, C.,… More

Segui, F., Daher, B., Gotorbe, C., Pouyssegur, J., Picco, V., Vucetic, M. Revealing the Ferroptotic Phenotype of Medulloblastoma. J. Vis. Exp. (205), e66645, doi:10.3791/66645 (2024).

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