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. 2020 May 14;11:843. doi: 10.3389/fimmu.2020.00843

FIGURE 2.

FIGURE 2

rSAA1 treated with RP-HPLC elution solvents retains its biological activity. (A) The chemotactic activity of rSAA1 treated with 50% acetonitrile and 0.1% trifluoroacetic acid (ST rSAA1) was evaluated on human neutrophils in the Boyden microchamber assay. The chemotactic potencies are expressed as mean chemotactic index + SEM derived from three independent experiments. Control migration is indicated by a dashed line (—). (B) FPR2-transfected HEK293 cells were stimulated with rSAA1 (6000 ng/ml, 500 nM) treated with 50% acetonitrile and 0.1% trifluoroacetic acid. Afterward, the cells were stimulated with WKYMVm (10 ng/ml, 12 nM) as control. (C) FPR2-transfected HEK293 cells were stimulated with WKYMVm (10 ng/ml, 12 nM). (D) FPR1-transfected HEK293 cells were stimulated with rSAA1 (6000 ng/ml, 500 nM). Afterward, the cells were stimulated with fMLF (10–10 M) as control. (E) FPR1-transfected HEK293 cells were stimulated with fMLF (10–10 M). (B–E) Changes in intracellular calcium levels were monitored by spectrophotometry. Results are presented as the ratio of emission of calcium-bound fura over calcium-free fura. One representative experiment out of two independent experiments is shown.