Ab133995

hMDMs were treated with DMSO or 50 nM DPI for 24 h. The culture supernatant was collected and an equal amount from four donors was mixed and applied to the human cytokine antibody array (#AB169827, Abcam) according to the manufacturer’s manual. The array has a panel of antibodies to detect 60 human cytokines or chemokines with duplicate spots for each on the film. The films were scanned by ImageQuant LAS 4000 (Cytiva) with the same exposure time. To examine whether DPI induces inflammation in vivo, wildtype B6 mice were dosed with 2 mg/kg DPI i.p. and plasma was collected before dosing and 48 h after dosing. An equal amount of plasma from four mice was mixed and applied to the mouse cytokine antibody array (#AB133995, Abcam) according to the manufacturer’s manual. The array has a panel of antibodies to detect 62 mouse cytokines or chemokines with duplicate spots for each on the film. The films were scanned by ImageQuant LAS 4000 (Cytiva) with the same exposure time.

A mouse cytokine array was used for simultaneous detection of 62 cytokines according to the manufacturer’s protocol (ab133995, Abcam, Cambridge, MA, USA). Briefly, mouse peritoneal cells were lysed in cell lysis buffer comprising 0.1 M Tris (pH 7.6) containing 0.15 M NaCl and 0.5% Nonidet P-40. The cell lysate was then added to the membrane of a mouse cytokine array. After washing the membrane, the detection antibody was applied and immunoblot images were captured using the BioSpectrum Imaging System. The intensity of each spot was measured using Image J software (version 1.44, NIH, Maryland, USA).

Primary microglia were isolated from mixed glial cultures from wild-type and P2ry6^−/− mice, and treated with ± 100 ng/ml lipopolysaccharide for 16 h, then the extracellular cell supernatant was centrifuged at 10,000 RCF to remove cellular debris. Supernatant was then assayed using an ELISA for 62 mouse cytokines and chemokines as per the manufacturer’s instructions (abcam, ab133995). Densitometric measurements were quantified using ImageJ, whereby intensity values were normalised between membranes using positive control spots. The fold change ± LPS for P2ry6^+/+ and P2ry6^−/− microglia was tested for significance using unpaired t tests for each cytokine/chemokine, followed by a Holm–Šídák multiple comparisons test. There were no significant differences for any cytokine/chemokine: VEGF, VCAM-1, thrombopoietin, sTNF RI, sTNF RII, TNF α, TIMP-1, TECK, TCA-3, TARC, SDF-1 α, SCF, RANTES, P-selectin, PF-4, MIP-3 α, MIP-3 β, MIP-2, MIP-1 γ, MIP-1 α, MIG, M-CSF, MCP-5, MCP1, lymphotactin, L-selectin, LIX, leptin, leptin R, KC, IL-17, IL-13, IL-12 p70, IL-12 p40/70, IL-10, IL-9, IL-6, IL-4, IL-3 Rb, IL-3, IL-2, IL-1β, IL-1α, IGFBP-6, IGFBP-5, IGFBP-3, IFN-γ, GM-CSF, GCSF, fractalkine, Fas ligand, eotaxin-2, eotaxin, CXCL16, CTACK, CRG-2, CD40, CD30 T, CD30 L, BLC and Axl (based on three independent experiments).