human microglia hmc3 cell line  (ATCC)

Journal: bioRxiv

Article Title: Modelling ferroptosis in a human microglial line by sequential exposure to iron and GPX4 inhibition

doi: 10.64898/2026.01.19.700282

Figure Lengend Snippet: A) Dose-response curves with RSL3 treatment (0-1 µM) for 2 hr with measurement of (i) levels of lipid peroxidation (BODIPY 581/591 C11) and (ii) cell viability (DRAQ7). Dotted line represents control values from DMSO-treated samples against which other samples are compared for statistical significance. N=3, one-way ANOVA with Šidák’s test, *p < 0.05, ***p < 0.001. B) Cytofluorimetric measurement of (i) levels of lipid peroxidation and of (ii) cell viability, following treatment with Erastin (10 µM), RSL3 (200 nM) or DMSO (control). N=3, one-way ANOVA with Tukey’s test, **p < 0.005, ***p < 0.001. C) (i) Overview of the experimental design. Dose-response curves of iron-loading (0-500 µM) for 24 hr, followed by treatment with DMSO (circles) or RSL3 (200 nM, triangles) for 2 hr measuring levels of (ii) lipid peroxidation and (iii) cell viability. Dotted lines represent control values from samples without iron against which the other samples are compared for statistical significance. N=3, one-way ANOVA with Dunett’s test, *p < 0.05. D) Rescue phenotype with Fer-1 (10 µM) after single or combined treatments of iron (50 µM) and RSL3 (200 nM) measuring levels of (i) lipid peroxidation and (i) cell viability. N=3, one-way ANOVA with Tukey’s test, *p < 0.05, ***p < 0.001. E) Representative images of HMC3 cells stained with (i) CC3 antibody (Green). Menadione treatment was used as positive control to induce apoptosis. (ii) Quantification of CC3 positive nuclei, plotted as percentage of total number of nuclei defined with Hoechst. Scale bar represents 100 µm. (iii) HMC3 survival measuring total nuclei (Hoechst) and plotted as percentage over DMSO-treated control. N=3, one-way ANOVA with Tukey’s test, **p < 0.01.

Article Snippet: Human microglia HMC3 cell line was purchased from ATCC (CRL-3304) and cultured in MEM containing Earl’s Salts and glutamine (Gibco, 31095-029) supplemented with 10% FBS (Gibco, 16140-063), 1% penicillin and streptomycin (Gibco, 15140-122) at 37°C and in a humidified atmosphere containing 5% CO2.

Techniques: Control, Staining, Positive Control

Journal: bioRxiv

Article Title: Modelling ferroptosis in a human microglial line by sequential exposure to iron and GPX4 inhibition

doi: 10.64898/2026.01.19.700282

Figure Lengend Snippet: A) (i) Representative images of HMC3 cells stained with CellMask Green and CellRox Orange. The staining was performed after 24 hr iron-loading of the cells (50 µM FAC) followed by treatment for 2 hr with DMSO or RSL3 (200 nM) +/- Fer-1 (10 µM). Scale bar=100µm. (ii) Quantification of CellRox intensity for DMSO (grey) or RSL3 (orange). N=3, one-way ANOVA with Šidák’s test, ***p < 0.001. B) MitoSOX labelling and quantification using flow cytometry to detect (i) mitochondrial superoxide and (ii) total ROS. N=4, one-way ANOVA with Šidák’s test, **p < 0.00. C) Workflow of cell culture for the model and cell painting analysis. Scale bar=100µm. D) Contingency tables listing the numbers of most important features per organelle/compartment in the top 35-40 features, ranked by Random Forest classifier, to differentiate treatment conditions. Cell summaries were excluded from Fisher Exact Test as too few of these features were present.

Article Snippet: Human microglia HMC3 cell line was purchased from ATCC (CRL-3304) and cultured in MEM containing Earl’s Salts and glutamine (Gibco, 31095-029) supplemented with 10% FBS (Gibco, 16140-063), 1% penicillin and streptomycin (Gibco, 15140-122) at 37°C and in a humidified atmosphere containing 5% CO2.

Techniques: Staining, Flow Cytometry, Cell Culture

Journal: bioRxiv

Article Title: Modelling ferroptosis in a human microglial line by sequential exposure to iron and GPX4 inhibition

doi: 10.64898/2026.01.19.700282

Figure Lengend Snippet: A) Overview of sample collection process. B) Differentially abundant lipids represented on Volcano plots for all the treatments. The top 10 significant lipids, ranked by adj. p-value and log2 fold-change are highlighted alongside most significant sterols. C) Heatmap showing abundance of lipids averaged across replicates and LipidMaps classes. Lipid abundance data is on a unit-variance scale to make analytes comparable with each other. D) Lipid enrichment analysis barplot showing top 10 lipid classes upregulated in ferroptotic HMC3 (positive log2 fold change of IronRSL3 vs DMSO). Sub-classes obtained from LipidMaps. MWU test to identify differential lipids (p <0.05) and ORA performed on lipid classes). E) Barplots showing top 20 (i) LipidMaps classes and (ii) lipid species restored by Fer-1 treatment (negative log2 fold change of IronRSL3-Fer1 vs IronRSL3).

Article Snippet: Human microglia HMC3 cell line was purchased from ATCC (CRL-3304) and cultured in MEM containing Earl’s Salts and glutamine (Gibco, 31095-029) supplemented with 10% FBS (Gibco, 16140-063), 1% penicillin and streptomycin (Gibco, 15140-122) at 37°C and in a humidified atmosphere containing 5% CO2.

Techniques:

Journal: iScience

Article Title: Spatial single-cell profiling identifies protein kinase Cδ-expressing microglia with anti-tumor function in glioblastoma

doi: 10.1016/j.isci.2025.114281

Figure Lengend Snippet: PKCδ in microglia contributes to the phagocytosis of BTICs (A) Schematic overview of the in vitro phagocytosis assay. (B and C) Representative IF images (B) and quantification (C) of phagocytosis of pHrodo-labeled S. aureus BioParticles by HMC3 microglia cells with PRKCD knockdown, in the presence or absence of niacin. (D) Phagocytosis assay using primary human microglia stimulated with niacin, with or without the PKC inhibitor CRT0066101. (E) Schematic of the in vitro phagocytosis assay using human BTICs labeled with pHrodo. (F and G) Representative images (F) and quantification (G) of phagocytosis of pHrodo-labeled human BTICs (BT012) by HMC3 cells with PRKCD knockdown. (H and I) Representative IF images (H) and quantification (I) of apoptotic BTICs (BT012 and BT025), determined by activated caspase-3/7 staining following co-culture with control or PRKCD -knockdown HMC3 cells, in the presence or absence of niacin. (J) Cell-type deconvolution of Visium spatial transcriptomics data from tumor-bearing mice treated with niacin. (K) Comparison of Prkcd expression between niacin-treated and control mice in spatial transcriptomics. (L) Quantification of Prkcd expression across spatial clusters. (M) IF staining of PKCδ and IBA1 in brain sections from vehicle- and niacin-treated mice. Statistical comparisons among multiple treatment groups were conducted using one-way ANOVA followed by Benjamini-Hochberg correction. Differences in Prkcd expression between spatial slides were assessed using the Wilcoxon rank-sum test ( p < 0.05). Data in (C and D), and I are presented as mean ± SEM. Scale bars on IF images: 50 μm.

Article Snippet: The human microglia cell line HMC3 (ATCC CRL-3304) was used for in-vitro validation experiments, including PRKCD knock-down assays.

Techniques: In Vitro, Phagocytosis Assay, Labeling, Knockdown, Staining, Co-Culture Assay, Control, Comparison, Expressing