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 undergo morphology changes in response to various pro-inflammatory stimuli. Representative phase-contrast images of unstimulated HAPI cells (control) and HAPI cells following 18 hours of stimulation with the toll-like receptor 4 agonist lipopolysaccharide (LPS, 100 ng/mL), the type I interferon, interferon-alpha (IFN-α, 10 ng/mL), the type II interferon, interferon-gamma (IFN-γ, 10 ng/mL), or combined LPS/IFN-α or LPS/IFN-γ at the same concentrations. The white arrowheads denote examples of protrusions with phagocytic cups.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control

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 cellular bioenergetic function is altered by various pro-inflammatory stimuli. (A) Cell number of the non-activated control (CTRL) group plotted across the five experiments, showing the variability binned into low- and high-cell-density categories. (B) Average HAPI cell oxygen consumption rate (OCR) measurements normalized to cell number following 18 hours of stimulation with lipopolysaccharide (LPS, 100 ng/mL), interferon-gamma (IFN-γ, 10 ng/mL), interferon-alpha (IFN-α, 10 ng/mL), or combined LPS/IFN-γ or LPS/IFN-α at the same concentrations. The results are mean ± SEM of n=5 biological replicates, with each experiment consisting of 2-3 technical replicates. The serial additions of the uncoupler FCCP (4 µM) + pyruvate (pyr, 10 mM), the nitric oxide scavenger cPTIO (200 μM), and the Complex III inhibitor antimycin A (Anti A, 1 μM) are indicated by arrows. (C) Basal mitochondrial OCR normalized to cell number calculated from the data shown in B. (D) Basal extracellular acidification rate (ECAR) normalized to cell number. ECARs were acquired simultaneously to OCR for the experiment shown in B. (E) Maximal mitochondrial OCR normalized to cell number calculated from the data shown in B. The data in panels C-E were analyzed by two-way analysis of variance (ANOVA) with treatment and experiment number as factors, followed by Tukey’s post hoc analysis. Experiment number had a significant effect on basal OCR (p<0.05) and basal ECAR (p<0.0001), while only a strong trend was observed on maximal OCR (p=0.06). (F) OCRs before and after cPTIO addition for the indicated treatment groups for the experiment shown in B. The data in panel F were analyzed by two-way ANOVA with repeated measures and Šídák’s multiple comparison’s test. (G) Linear regression analysis of cPTIO-induced OCR versus cell number for the LPS/IFN-γ and LPS/IFN-α treatment groups, with R 2 and p-values indicated on the graph. The p-values indicate the probability of a non-zero slope of the linear fits. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control

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: Eighteen-hour activation of HAPI cells with lipopolysaccharide plus interferon-gamma (LPS/IFN-γ) or LPS plus interferon-alpha (LPS/IFN-α) reduces cell number compared to the non-activated control (CTRL) group. HAPI cells were treated with the following stimuli prior to the determination of cell number by manual cell counting of three representative fields per treatment: LPS (100 ng/mL), IFN-α (10 ng/mL), IFN-γ (10 ng/mL), LPS/IFN-γ (100 ng/mL / 10 ng/mL, respectively), or LPS/IFN-α (100 ng/mL / 10 ng/mL, respectively). Cell number is shown in (A) while the results in (B) express the cell number as a percentage of the CTRL group mean. The results are mean ± SEM of n=5 biological replicates. The data were analyzed by two-way analysis of variance with treatment and experiment number as factors, followed by Tukey’s post hoc analysis. Both the treatment and the experiment number had a significant effect (p<0.0001 each), and there was a significant interaction between the two (p<0.05). ***p<0.001, ****p<0.0001.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Activation Assay, Control, Cell Counting

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: Respiratory suppression induced by combined lipopolysaccharide/interferon-gamma (LPS/IFN-γ) stimulation is partly attenuated by an inhibitor of inducible nitric oxide synthase (iNOS). (A) HAPI cell oxygen consumption rate (OCR) measurements from an unstimulated control (CTRL) group or following 18 hours of stimulation with LPS/IFN-γ (100 ng/mL / 10 ng/mL, respectively) in the absence or presence of the iNOS inhibitor 1400W (50 µM). The serial additions of FCCP (4 µM) + pyruvate (pyr, 10 mM), cPTIO (200 μM), and antimycin A (Anti A, 1 μM) are indicated by arrows. (B) The data in A expressed as a percentage of the baseline OCR (third measurement) because cell counts were not done for this experiment. Baseline normalization removes the influence of cell number on OCR, illustrating that the recovery of respiratory capacity is incomplete. The results are mean ± SD of n=3 technical replicates and are representative of n=7 similar experiments done in triplicate. Because this experiment was done as part of another study with additional comparisons, only the representative trace is shown, and the results will be reported in more detail elsewhere.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control