Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization

Table 1

N9 microglia exposed to LPS show increased cell death as indicated by Guava Nexin assay.

	Viable cells (%)	Early apoptosis (%)	Late apoptosis/necrosis (%)
Control	84 ± 8.3	10.6 ± 4.2	5.0 ± 3.0
LPS	66.9 ± 4.5	23.2 ± 7.6	10.3 ± 5.0

Phycoerythrin-conjugated annexin V (V-PE) and 7-aminoactinomycin D (7-AAD) (Guava Nexin Reagent, #4500-0450, Millipore) were used to determine the percentage of viable, early-apoptotic, and late-apoptotic/necrotic cells by flow cytometry. After incubation, cells were trypsinized, added to extracellular media, and then stained with annexin V-PE and 7-AAD. Samples were analyzed on a Guava easyCyte 5HT flow cytometer (Guava Nexin Software module, Millipore). Three distinct populations of cells were identified: viable cells (annexin V-PE and 7-AAD negative), early-apoptotic cells (annexin V-PE positive and 7-AAD negative), and late stages of apoptosis/necrosis (annexin V-PE and 7-AAD positive). versus nontreated cells (control).