Multiplex magnetic bead immunoassays

SASP factors: CCL‐2/MCP‐1, IL1α, MMP‐1, PAI1, TNFα, IL‐6, and IL‐10 (Basisty et al., 2020 (link); Camell et al., 2021 (link); Englund et al., 2021 (link); Huang et al., 2018 (link)). CCL‐2/MCP‐1, IL1α, MMP‐1, PAI1, and cystatin C were quantified using multiplex magnetic bead immunoassays (R&D Systems) based on Luminex xMAP multianalyte profiling platform (Luminex® 100/200™ System) according to the manufacturer's protocol and analyzed on Bio‐Rad Bio‐Plex Manager 6.1. Cytokine biomarkers analyzed included C‐reactive protein (CRP), tumor necrosis factor alpha (TNFα), and its receptors (TNFα‐R1 and TNFα‐R2), interleukin 6 soluble receptor (sIL‐6R), and interleukin 10 (IL‐10) as previously described (Langmann et al., 2017 (link)). For all proteins, more than 80% of the samples were within the detectable range. Undetectable targets were replaced with the lowest value for each protein.

Conditioned medium was collected from either non‐senescent or senescent FAPs in DMEM 4.5 g/l glucose supplemented with 10% FBS and 1% penicillin–streptomycin‐glutamine at 37°C at 3% O₂ incubator for 24 h. Conditioned medium was filtered through 0.22 μm filter before analysis, and the cell number was quantified using an automated cell counter (Biorad) for normalization. The concentrations of GDF‐15, TNFRI, TNFRII, IL‐6, IL‐10, ICAM‐1, PAI‐1, Eotaxin, MCP‐1, MMP‐2, and OPN in 50 μL of conditioned medium were quantified using multiplex magnetic bead immunoassays (R&D Systems).

The concentrations of CCL2, CCL3, CCL7, CCL8, CCL22, CXCL1, CXCL2, G-CSF, GDF-15, ICAM-1, IGFBP3, IL-1α, IL-1β, IL-7, IL-17, LIX, M-CSF, MMP-3, OPN, TNFSF6, TNFRSF1A, TNF-α, and VEGF in plasma were quantified using commercially available multiplex magnetic bead immunoassays (R&D Systems) based on the Luminex xMAP multianalyte profiling platform and analyzed on a MAGPIX System (Merck Millipore). All assays were performed according to the manufacturer’s protocols. Activin A and TGF-β1 concentration were determined by a Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Investigators were blinded as to sample identity and treatment of mice.