Monolayer cultures (20,000 cells/well in 8-well chambered slide) were washed with PBS, fixed with 4% paraformaldehyde (Thermo Fisher, Waltham, MA, USA), and blocked in PBS with 1% BSA (Sigma-Aldrich, Oakville, ON, Canada), 1% goat serum, and 0.1% Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada) for 1 h. Pam2CSK4 treated cells were stained with primary antibodies specific to NGF (Santa Cruz, Dallas, TX, USA), p16Ink4a (Cintec-Roche, Laval, Qc, CAN), IL1β, TNF-α, IL8, and TLR-2 (Abcam, Cambridge, Ma, USA) overnight at 4 °C. Healthy cells were treated with p16INK4a and TLR-2 only. After washing, cells were incubated with the appropriate Alexa Fluor® 488 or 594-conjugated secondary antibody (Thermo Fisher, Waltham, MA, USA) for 2 h at room temperature, and then counterstained with DAPI for nuclear staining. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan). Ten images of each condition per donor were analyzed and positive cell percentage was quantified by Fiji ImageJ (version: 2.1.0/1.53 c). Briefly, the number of cells stained positive for one of the target proteins (NGF, IL-1β, TNF-α, and IL-8) were counted and compared to the total number of cells positive for DAPI staining. For the double staining (TLR-2 and p16INK4a), the percentage of positive cells represents the ratio of the number of cells positively stained for either one of the 2 markers (TLR-2 and p16INK4a) divided by the total number of cells positively stained for DAPI.

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