human microglial cells  (ATCC)

Journal: bioRxiv

Article Title: Protein folding stress shapes microglial phenotype in progressive supranuclear palsy

doi: 10.64898/2026.01.26.700084

Figure Lengend Snippet: Changes in Human microglia cell (HMC3) Associated with ER Stress (A) Schematic graphs of in vitro assay. Created in BioRender. (B) Effects of tunicamycin on HMC3 cell numbers. Cell numbers were quantified with a Countess automated cell counter 24 h and 48 h after treatment with DMSO or the indicated concentrations of tunicamycin (n = 3 per condition). Bars represent mean ± SEM. Asterisks denote significant differences from the DMSO control at the corresponding time point, based on a linear model including Time and Treatment with Dunnett-adjusted contrasts versus the DMSO control (**p < 0.01, ***p < 0.001, ****p < 0.0001). (C) Effects of tunicamycin on LDH release. LDH activity in culture supernatants was measured 24 h and 48 h after treatment, and relative luminescence units (RLU) were normalized to cell number in each well (n = 3). Bars indicate mean ± SEM. Asterisks indicate significant differences from the DMSO control at the same time point, assessed using the same Dunnett-adjusted linear model (*p < 0.05, ****p < 0.0001). (D) Quantitative PCR analysis of protein folding / ER stress–related genes. Relative mRNA levels of ATF4 , GADD34 , HSP90B1 and HSPA1 in HMC3 cells treated with DMSO 48 h or tunicamycin 0.1 µg/mL for 48 h (n = 3; mean ± SEM). Two-sided Welch’s t-test were performed for each gene, and p values were adjusted for multiple testing across genes using the Benjamini–Hochberg correction (**adj. p < 0.01, ***adj. p < 0.001). (E) qPCR analysis of homeostatic microglial genes CX3CR1 and P2RY12 in HMC3 cells after 48 h treatment with DMSO, tunicamycin (0.05 or 0.1 µg/mL), or thapsigargin (1 µM) (n = 3 per condition). Data are shown as relative quantification normalized to the DMSO control (mean ± SEM). Asterisks indicate pairwise differences from the DMSO control based on two-sided Welch’s t-test with Benjamini–Hochberg correction for multiple comparisons (*adj. p < 0.05, **adj. p < 0.01). (F) Bright-field images of HMC3 cells treated with DMSO or tunicamycin (0.01, 0.05, 0.1 µg/mL) and thapsigargin (1 µM) for 24 h or 48 h. Scale bars, 100 µm. (G) Representative fluorescence images of HMC3 cells stained for HLA-DR/DP/DQ (green) and DAPI (blue) after 48 h treatment with DMSO, tunicamycin (0.1 µg/mL), or thapsigargin (1 µM). Scale bars, 20 µm. (H) Quantification of HLA-DR/DP/DQ–positive cell body area in HMC3 cells. Cell body area (µm²) was measured from thresholded HLA-DR/DP/DQ–positive regions in Fiji. Each point represents one field of view (5 fields per dish, 3 independent dishes per condition); boxes indicate the interquartile range with median, and whiskers show the range. Group differences among DMSO, tunicamycin (TM) and thapsigargin (THA) were assessed by one-way ANOVA followed by Tukey’s multiple comparison test (****p < 0.0001).

Article Snippet: HMC3 human microglial cells (ATCC, Manassas, VA, USA; CRL-3304) were cultured in DMEM, high glucose (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; 11965-092) with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified 5% COl incubator.

Techniques: In Vitro, Control, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Fluorescence, Staining, Comparison

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: HMC3 – Human microglial cell line , ATCC , CRL-3304.

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