Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Graphene‐dependent activation of SIM‐A9 microglia. (A) ATP content of SIM‐A9 cells cultured for 3 h on glass or PET surfaces with (+) or without (−) monolayer graphene‐coating in PBS. (B) Representative images of cells on glass (Gl) and graphene‐coated glass (Gr/Gl); scale bar: 200 µm. (C) TNF‐ α secretion measured by ELISA. Lipopolysaccharide (LPS) treatment served as a positive control. (D) Cells were cotreated with surfaces or LPS in combination with polymyxin B for 3 h. Data are presented as percentage of control (untreated cells on tissue culture plastic) and shown as mean ± SD from at least two independent experiments ( n ≥ 3 each). Statistical analysis was performed using one‐way ANOVA with Sidak's post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Positive Control, Control

Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Multilayering does not affect graphene‐induced activation of SIM‐A9 cells. (A) Raman spectra of glass substrates (Gl) coated with 1–3 layers of graphene (1×–3× Gr/Gl); exemplary spectra from single points are shown. (B) Contact angle measurements ( n = 3 per group). (C,D) Atomic force microscopy (AFM) images of graphene surfaces; representative images are shown ( n = 5 per group). (E) ATP content of SIM‐A9 cells cultured for 3 h on glass coated with 1–3 layers of graphene. Data are presented as percentage of control and shown as mean ± SD from three independent experiments ( n = 3 per group). Statistical analysis was performed using one‐way ANOVA with Sidak's post hoc test (ns, p > 0.05; * p < 0.05; *** p < 0.001).

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Activation Assay, Microscopy, Cell Culture, Control

Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Carbon nanotubes (NT) but not nanoplatelets (NP) induce stimulation of SIM‐A9 cells. (A) ATP content of SIM‐A9 cells incubated for 3 h with graphene nanomaterials (amount adjusted to match a graphene monolayer). (B) TNF‐ α secretion measured by ELISA; lipopolysaccharide (LPS) served as a positive control. (C,D) Prostaglandin and nitric oxide levels measured in cell supernatants. Data are presented as percentage of control and shown as mean ± SD from three independent experiments ( n = 2 each). Statistical analysis was performed using Kruskal–Wallis test with Dunn's multiple comparisons (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Positive Control, Control

Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Uptake of graphene nanomaterials and morphological changes in SIM‐A9 cells after 24 h of exposure. (A) Representative microscopic images of cells incubated with graphene nanoplatelets (NP) or nanotubes (NT) in serum‐free medium for 24 h; serum‐free medium alone served as control. (B) Percentage of cells containing ingested particles (mean ± SD); quantified from all evaluated cells. Statistical analysis: unpaired t ‐test (* p < 0.05). (C) Size distribution of ingested particles, assessed using the Single Cellome System SS2000. (D) Morphological alterations in SIM‐A9 cells, including blebbing (upper, indicated by white arrow) and mitochondrial fragmentation (lower; mitochondria stained with LumiTracker Red). Percentage of damaged cells (cells were designated “damaged” when showing blebbing and/or mitochondrial defects) presented as mean ± SD. Cells were evaluated in three independent experiments ( n = 8). Statistical analysis: one‐way ANOVA with Sidak's multiple comparisons (*** p < 0.001; ** p < 0.01). Scale bar: 10 µm.

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Incubation, Control, Staining

Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Raman spectra of previously graphene‐coated glass after colonization with SIM‐A9 cells. Graphene‐coated glass was incubated with SIM‐A9 cells for 3 h. (A) Raman spectra were recorded from a 20 × 20 µm 2 area at the center of the sample before and after cell culture (six independent samples). Exemplary point spectra from one sample each are shown. (B) Quantification of points showing graphene‐derived Raman signals before and after cell contact. Data are presented as mean ± SD. Statistical analysis: unpaired t ‐test (*** p < 0.001).

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Incubation, Cell Culture, Derivative Assay

Journal: Small Science

Article Title: Graphene Triggers Inflammation in Murine Microglia via Phagocytosis

doi: 10.1002/smsc.202500531

Figure Lengend Snippet: Identification of molecular pathways involved in graphene‐based microglial stimulation via proteome analysis. Proteomic analysis was performed on SIM‐A9 cell lysates after 3‐h incubation with graphene on glass (Gl/Gl), graphene nanoplatelets (NP), or carbon nanotubes (NT) in PBS, compared to cells on pure glass. Only proteins with log 2 fold change >1.5 or <−1.5 and adjusted p ‐value < 0.05 were considered. (A) Volcano plot showing differential protein expression relative to glass control. The x ‐axis represents log 2 fold change, and the y ‐axis represents ‐log 10 ( p ‐value) from a modified t‐statistic (t(SAM)). (B) Venn diagrams showing overlap of significantly upregulated (blue, left) and downregulated (red, right) proteins. Circle size does not correspond to protein quantity. (C) Gene ontology analysis of up‐ and downregulated proteins, highlighting the 10 most strongly regulated pathways. Color scale indicates pathway strength: blue = upregulation, red = downregulation. (D,E) Protein–protein interaction networks of the top 50 significantly upregulated (D) and downregulated (E) proteins, visualized using STRING. Nodes represent proteins; edges represent predicted functional associations, with edge thickness reflecting interaction confidence. Three central networks were identified for both up‐ and downregulated proteins using k‐means clustering (minimum interaction score: 0.400). White spheres indicate unclustered proteins. Proteomics were performed with n = 4 per condition from four independent experiments.

Article Snippet: SIM‐A9 cells (Applied Biological Materials Inc.; Richmond (BC), Canada) were cultured at 37°C, 95% humidity, and 5% CO 2 using Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 (DMEM:F‐12), supplemented with 10% v/v heat‐inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 5% v/v heat‐inactivated donor horse serum (Gibco, Carlsbad, CA, USA), 1% v/v Penicillin–Streptomycin (Sigma–Aldrich; Steinheim, Germany), and 1% v/v L‐Glutamine (Sigma–Aldrich; Steinheim, Germany).

Techniques: Incubation, Expressing, Control, Modification, Functional Assay

Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet:

Article Snippet: The number of repeats varies greatly among cell lines, making STRs highly useful for identification by providing a cell line “fingerprint.” STR profiling revealed that HAPI cells are a 100% STR match for SIM-A9 cells, a mouse microglial cell line in the ATCC database that was reported to originate from a postnatal day 1 primary mouse microglial culture through spontaneous immortalization ( ) ( ; Figure S1).

Techniques:

Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet: HAPI cells express microglia-selective markers. (A) Representative image of HAPI cells immunostained for TMEM119. (B) Detection of Tmem119 polymerase chain reaction (PCR) product in sample prepared from HAPI cells, SIM-A9 cells, mouse cortex tissue, mouse bone marrow-derived macrophages (BMDMs), or Neuro2a cells. An arrow designates the band corresponding to the predicted product size of 119 base pairs (bp). (C) Detection of Cx3cr1 PCR product in the same samples evaluated in B. An arrow designates the band corresponding to the predicted 109 bp product.

Article Snippet: The number of repeats varies greatly among cell lines, making STRs highly useful for identification by providing a cell line “fingerprint.” STR profiling revealed that HAPI cells are a 100% STR match for SIM-A9 cells, a mouse microglial cell line in the ATCC database that was reported to originate from a postnatal day 1 primary mouse microglial culture through spontaneous immortalization ( ) ( ; Figure S1).

Techniques: Polymerase Chain Reaction, Derivative Assay