Functional comparison of BMDMs differentiated under different plating densities. (a) TNF-α, IL-6, IL-12, KC and MIP-1α were measured from culture supernatant using ELISA kits from Peprotech following the manufacturer’s instructions. Cells were plated on 6-well plates and stimulated with 100 ng/mL LPS (Escherichia coli serotype 0128:B12, Sigma) for 18 hours. (b) Phagocytosis assay was performed by plating 1x105 cells on a 96-well plate in triplicates and incubating with fluorescein-tagged K-12 bioparticle (Invitrogen) for two hours and nonspecific signals were blocked with trypan blue. Fluorescence was detected on the Molecular Devices Spectra Max 190 microplate reader at 480 nm excitation and 520 nm emission wavelengths. (c) Presence of nitric oxide (NO) in the culture supernatant of the LPS-stimulated BMDM was measured colometrically using Greiss reagent (Molecular Probes) following the manufacturer’s instructions. Results represent three experiments of three biological samples each. Statistical analysis was done by one-way ANOVA with Bonferroni post-test. *** P < 0.0005; ** P < 0.005; * P < 0.05.