human microglia primary cell culture complete media with serum  (Celprogen Inc)

Journal: Neural Regeneration Research

Article Title: Mesenchymal stem cell–derived small extracellular vesicles enhance the therapeutic effect of retinal progenitor cells in retinal degenerative disease rats

doi: 10.4103/NRR.NRR-D-23-02108

Figure Lengend Snippet: Microglial suppression of RCS rats mediated by co-transplantation of RPCs with M-sEVs. (A) Representative confocal images of Iba1 staining in the RPCs and RPCs + M-sEVs groups at 2 and 4 weeks post transplantation. Enlarged orthogonal view of M-sEVs at the injected area (right panel) shows co-localization of the M-sEVs with microglia in the subretinal space. (B) Statistical analysis of the number of Iba1-positive cells in each group in A. (C, D) Representative confocal images showing the localization of MSC-sEVs (PKH26 staining) with F-actin-labeled BV2 (C) and HMC3 (D) microglial cells stimulated with LPS. Scale bars: 20 μm (A), 10 μm (A, enlarged orthogonal view), 50 μm (C, D). (E) RT-PCR analysis showing relative mRNA expression levels of IL-1β, IL-6, and TNF-α in the mouse (BV2) and human (HMC3) microglial cell lines. Data are expressed as means ± SD ( n ≥ 3 for Iba1 staining; n = 3 for real-time PCR); * P < 0.05, ** P < 0.01, *** P < 0.001 (B: Welch’s t -test; D: Welch’s one-way analysis of variance followed by Dunnett’s T3 multiple-comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; EGFP: enhanced green fluorescent protein; Iba1: ionized calcium-binding adapter molecule 1; IL-1β; interleukin 1 beta; IL-6: interleukin 6; LPS: lipopolysaccharide; M-sEVs: mesenchymal stem cell–small extracellular vesicles; RT-PCR: real-time polymerase chain reaction; RPCs: retinal progenitor cells; RCS: Royal College of Surgeons; TNF-α: tumor necrosis factor α; w: weeks.

Article Snippet: Human microglia clone 3 (HMC3) cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China, Cat# CL-0620, RRID: CVCL_II76) and were cultured in alpha minimum essential medium (Cat# SH30265.01, Hyclone) containing 10% FBS.

Techniques: Transplantation Assay, Staining, Injection, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Comparison, Binding Assay

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

Journal: Bioactive Materials

Article Title: Dimensional memory in glioblastoma mechanics: Traction force analysis of cells cultured in 2D versus 3D collagen environments

doi: 10.1016/j.bioactmat.2025.09.025

Figure Lengend Snippet: A schematic of experimental design including representation of GB tumor microenvironment where glioblastoma cells also interact with other cell types, i.e., microglia and astrocytes, etc. This research includes glioblastoma cell lines (T98G and LN229) and microglia cell line HMC3, which were set in two pre-culture conditions, i.e., 2D and 3D, to perform a cell proliferation assay further followed by single-cell traction force microscopy, immunofluorescence assays, and single-cell nanoindentation studies .

Article Snippet: Human-derived malignant GB cells LN229 (ATCC-CRL-2611, Germany), T98G (ATCC-CRL-1690, Germany), and human microglia cells HMC3 (ATCC CRL-3304, Germany) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % v/v fetal bovine serum (FBS, PAN-Biotech Gmb, Aidenbach, Germany), 1 % penicillin-streptomycin (Sigma Life science, St. Louis, USA), 1.0 % v/v Glutamax (Gibco, New York, US) and 0.1 % v/v Gentamicin (Gibco, New York, US) in T75 culture flasks (SARSTEDT AG & Co.KG, Nümbrecht, Germany).

Techniques: Proliferation Assay, Microscopy, Immunofluorescence

Journal: Bioactive Materials

Article Title: Dimensional memory in glioblastoma mechanics: Traction force analysis of cells cultured in 2D versus 3D collagen environments

doi: 10.1016/j.bioactmat.2025.09.025

Figure Lengend Snippet: Representative graphs of the cell proliferation assay of HMC3, LN229, and T98G. In these graphics, the gray curve represents the duplication of cells in the 3D condition, while the orange and blue curves depict the proliferation of cells under 2D conditions and control, respectively.

Article Snippet: Human-derived malignant GB cells LN229 (ATCC-CRL-2611, Germany), T98G (ATCC-CRL-1690, Germany), and human microglia cells HMC3 (ATCC CRL-3304, Germany) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % v/v fetal bovine serum (FBS, PAN-Biotech Gmb, Aidenbach, Germany), 1 % penicillin-streptomycin (Sigma Life science, St. Louis, USA), 1.0 % v/v Glutamax (Gibco, New York, US) and 0.1 % v/v Gentamicin (Gibco, New York, US) in T75 culture flasks (SARSTEDT AG & Co.KG, Nümbrecht, Germany).

Techniques: Proliferation Assay, Control

Journal: Bioactive Materials

Article Title: Dimensional memory in glioblastoma mechanics: Traction force analysis of cells cultured in 2D versus 3D collagen environments

doi: 10.1016/j.bioactmat.2025.09.025

Figure Lengend Snippet: (A) Illustration of the method to analyze traction stress exerted by single cells . This analytical strategy requires a flat polyacrylamide hydrogel functionalized with collagen type I. (B) The box plot represents the elastic properties of the aforenamed PAA hydrogel from one representative gel of three independently prepared PAA gels; data from all three gels are shown in , measured by nanoindentation, exhibiting an average elasticity of approximately 3.6 kPa (measured as Young's modulus). (C) Quantification of the total strain energy exerted by single cells after being extracted from 2D hard substrates and 3D soft substrates, later anchored to polyacrylamide hydrogels functionalized with collagen type I. (D) Determination of the F/A ratio for HMC3, LN229, and T98G. (E) Mean focal adhesion stress for each cell line in both culture conditions. Data represent pooled measurements from 5 to 10 independent experiments per condition, (n = 85–193) cells per condition. Mann-Whitney U Test was performed after random resampling for statistical analysis between two conditions for (C), (D), and (E), where ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Human-derived malignant GB cells LN229 (ATCC-CRL-2611, Germany), T98G (ATCC-CRL-1690, Germany), and human microglia cells HMC3 (ATCC CRL-3304, Germany) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % v/v fetal bovine serum (FBS, PAN-Biotech Gmb, Aidenbach, Germany), 1 % penicillin-streptomycin (Sigma Life science, St. Louis, USA), 1.0 % v/v Glutamax (Gibco, New York, US) and 0.1 % v/v Gentamicin (Gibco, New York, US) in T75 culture flasks (SARSTEDT AG & Co.KG, Nümbrecht, Germany).

Techniques: MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Dimensional memory in glioblastoma mechanics: Traction force analysis of cells cultured in 2D versus 3D collagen environments

doi: 10.1016/j.bioactmat.2025.09.025

Figure Lengend Snippet: Changes in the number of focal adhesions based on cytoskeleton images of HMC3, LN229, and T98G cells seeded on glass coverslips after culturing them on 2D collagen-based surfaces (A) and 3D collagen-based bioactive matrices (B). In these images, red staining indicates the presence of F-actin, while green staining displays focal adhesions (i.e., vinculin). Based on these images, cell area (C), the number of focal adhesions per cell (D), and the focal adhesion area (E) were calculated. In the last three images, blue bars correspond to cells pre-conditioned on 2D collagen-coated surfaces, while orange bars represent values obtained from cells cultured in 3D collagen-based bioactive matrices. Statistical analysis was performed using the Welch's t -test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. Significance levels: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: Human-derived malignant GB cells LN229 (ATCC-CRL-2611, Germany), T98G (ATCC-CRL-1690, Germany), and human microglia cells HMC3 (ATCC CRL-3304, Germany) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % v/v fetal bovine serum (FBS, PAN-Biotech Gmb, Aidenbach, Germany), 1 % penicillin-streptomycin (Sigma Life science, St. Louis, USA), 1.0 % v/v Glutamax (Gibco, New York, US) and 0.1 % v/v Gentamicin (Gibco, New York, US) in T75 culture flasks (SARSTEDT AG & Co.KG, Nümbrecht, Germany).

Techniques: Staining, Cell Culture, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Dimensional memory in glioblastoma mechanics: Traction force analysis of cells cultured in 2D versus 3D collagen environments

doi: 10.1016/j.bioactmat.2025.09.025

Figure Lengend Snippet: A) A schematic of the nanoindentation experiment is presented detailing indentation on single cells conducted using a cantilever with a spherical tip. B) Representative load–indentation curves featuring Hertz fits. C) Stiffness measurements obtained through the peak load poking mode of the nanoindenter on cells. The elasticity (Young's modulus) of HMC3, LN229, and T98G was analyzed using the single-cell nanoindentation method. Similar to other mechanical characterizations, these cell lines underwent specific culture pre-culture conditions, i.e., a 2D hard substrate and a 3D soft substrate, along with a conventional culture flask serving as the control. Mann-Whitney U Test was conducted for statistical analysis among the three conditions, where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Human-derived malignant GB cells LN229 (ATCC-CRL-2611, Germany), T98G (ATCC-CRL-1690, Germany), and human microglia cells HMC3 (ATCC CRL-3304, Germany) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % v/v fetal bovine serum (FBS, PAN-Biotech Gmb, Aidenbach, Germany), 1 % penicillin-streptomycin (Sigma Life science, St. Louis, USA), 1.0 % v/v Glutamax (Gibco, New York, US) and 0.1 % v/v Gentamicin (Gibco, New York, US) in T75 culture flasks (SARSTEDT AG & Co.KG, Nümbrecht, Germany).

Techniques: Control, MANN-WHITNEY