LPS-induced ICAM-1 expression in A549 cells

A549 cells (1.5 × 10⁵) were incubated in individual wells of a 24-well plate with coverslips placed in each well for 24 h. E. coli and Geitlerinema sp. LPS was initially diluted in PBS. Cells were treated with 100,000 ng/mL of E. coli LPS or 100,000 ng/mL of Geitlerinema LPS for 6 h. This concentration was used based on previous studies demonstrating a peak LPS response of immune cells in vitro [28 (link),29 (link)]. Cells were fixed with methanol at 4 °C overnight. Cells were washed three times with PBS-Tween (0.02%) and incubated in 5% normal goat serum for 1 h at room temperature to block nonspecific binding. After subsequent washing, cells were incubated at 4 °C overnight in primary antibodies to rabbit CD54/ICAM-1 (1:25 Cell Signaling, Danvers, MA, USA). Cells were washed of primary antibody, then incubated with anti-rabbit IgG (AlexaFluor 488, Cell Signaling #4412, Danvers, MA, USA) at room temperature for 1 h, washed again, and mounted in VectaShield HardSet™ with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained on a Nikon EclipseTi confocal microscope (Nikon Instruments, Melville, NY, USA). Image files were quantified by the MFI and mean gray value using imageJ software (National Institutes of Health (NIH), Bethesda, MD, USA). The experiment was performed independently three times.