Fig. 6: TNFα increases the number of TH+ monocytes and the amount of TH protein per monocyte.

a Total of CD14+ monocytes were isolated using a negative magnetic selection from 80 million healthy donor PBMCs. Monocytes were seeded into a six-well ultra-low-adherence plate at 2 million cells per well and treated with vehicle (media), TPA (100 ng/mL, positive control), TNFα (17 ng/mL), in duplicate. b One duplicate was assayed by flow cytometry to detect TH-expressing monocytes, using counting beads as a reference value to quantify the number of TH+ cells. c Number of TH+ cells were quantified as shown. d Representative histogram showing one set of samples assayed for TH-expressing monocytes following stimulation. e Both TPA and TNFα induced significant increases in TH-expressing monocytes relative to vehicle, shown as fold-increase relative to vehicle (n = 3 per group, one-way ANOVA, p < 0.01). f No increase in total monocytes per condition, relative to vehicle (n = 3 per group, one-way ANOVA, n.s.). g TH concentration in picograms per milligram total protein shows TNFΑ treatment results in significantly increased TH protein relative to vehicle and TPA (n = 5–6 per group, one-way ANOVA, p < 0.001). h Mean TH protein level for monocytes treated with vehicle, TPA and TNFα are plotted on a representative standard curve, with the inset magnifying the lower end of the curve. i Intracellular flow cytometry for Ki67 does not reveal significant differences between vehicle and TNFα treatment groups, confirming a lack of cell proliferation following TNFα treatment. Data are shown as ±SEM.