Fig 1: Neuronal characteristics and HIV co-receptor profile of H80 cells.(A) Immunofluorescence staining of H80 cells for NeuN using specific primary antibodies, with Hoechst (blue) counterstaining and unstained as controls. NeuN was detected, confirming the neuronal-like phenotype of H80 cells. (B) Comparative immunofluorescence staining of H80 cells, microglial cells, SH-SY5Y cells (positive control), and HEK293T cells (negative control) demonstrated that MAP2 expression was restricted to H80 and SH-SY5Y cells. The inclusion of HEK293T as a negative control and SH-SY5Y as a positive control confirmed assay specificity. Detection of MAP2 in H80 cells indicates that these cells exhibit features of differentiated neurons. (C) Immunoblotting verified MAP2 expression in H80 cells but not in HEK293T or microglial cells, further supporting the neuronal identity of H80 cells. Collectively, these findings demonstrate that H80 cells express canonical neuronal markers, including NeuN and MAP2, positioning them as a relevant model for investigating neuron-related molecular mechanisms, implicated in neurodegenerative disease and HIV/drug-induced neurotoxicity. (D) Flow cytometry analysis of HIV receptor expression on HEK293T, U937, and H80 cells. Cells were blocked with 2% BSA plus Fc block and co-stained for either CD4 and CXCR4 (APC anti-human CD4, BioLegend cat. no. 317416; PE anti-human CD184 \[CXCR4], BioLegend cat. no. 306505) or CD4 and CCR5 (PE anti-human CD4, BioLegend cat. no. 357403; APC/Cyanine7 anti-human CD195 \[CCR5], BioLegend cat. no. 359110). HEK293T cells (negative control) lacked CD4, CXCR4, and CCR5 expression, whereas U937 cells (positive control) expressed all three markers. H80 cells did not express CD4 or CCR5 but showed detectable CXCR4 expression. These findings suggest that H80 cells lack main HIV receptor CD4 and chemokine receptor CCR5 for HIV entry, despite expressing CXCR4.