Influx flow cytometer

All analytical flow cytometry was performed on a BD LSR II flow cytometer. Dead cells were identified by DAPI (4’,6-diamidino-2-phenylindole) and excluded from analysis where appropriate. Primary antibodies specific for the following markers were directly conjugated to FITC, PE, APC, PerCP-Cy5.5, eFluor-780, eFluor-450, or PE-Cy7 and purchased from eBioscience (CD3, CD4, CD8α, CD11b, CD16/32, CD34, CD45.1, CD45.2, CD45R(B220), CD49b(DX5), FcεR1 and Gr1) or from BD Biosciences (c-kit, IL-5Rα, Siglec F, CCR3, and Annexin V). For intracellular staining, surface-labeled cells were fixed in isotonic 3% paraformaldehyde and incubated with affinity-purified anti-CF (Hamilton et al., 2008 (link)) in Fix & Perm (Invitrogen) according to product instructions. Staining was detected with highly cross-adsorbed donkey anti-rabbit-FITC (Jackson Immunochemicals, Stratech, UK). For cell sorting experiments, eosinophil populations were sorted on a BD Influx flow cytometer.

UBI4 repeat variants carrying plasmids pYES-Ub-M-GFP (stable GFP) or pYES-Ub^G76V-GFP (ubiquitin fusion degradation substrate)^{27 (link), 28 (link)} (Addgene) were grown for two successive overnights at 30 °C in SC-URA medium containing 2% glucose and then 2% raffinose and 2% galactose. The cultures were next diluted in SC-URA medium containing 2% galactose and grown at 30 °C until midlog phase and then transferred to a water bath at 44 °C. At 0, 10, 30, 60, and 120 min after the temperature shift, 100 µl cultures were centrifuged and cells frozen in 25% glycerol until flow cytometry analysis. Fluorescence of 50,000 cells per sample was measured using a BD Influx flow cytometer equipped with a 488 nm laser and a 530/40 nm filter (GFP). We calculated the frequency of the remaining fluorescent cells over the total population at various time points using the FlowJo software (Tree Star, Inc.). Details on the calculation of the turnover rates are provided in Supplementary Information. A similar setup was used to check effect of proteasomal inhibition, where MG-132 (Sigma) was added to a final concentration of 100 µM.

Cells were plated in six-well plates with a density of 10⁵ per well for further detection. The intracellular ROS level was measured by ROS Assay Kit (Beyotime, China). The fluorescence intensity of DCFH-DA was read under a microplate reader and a BD Influx flow cytometer (BD Biosciences, USA), using excitation and emission wavelengths of 488 and 525 nm. The fluorescence images were observed by fluorescence microscopy and data was analyzed using FlowJo software. Lipid peroxidation levels reflect the extent of intracellular oxidative damage, as measured by Lipid Peroxidation MDA Assay Kit (Beyotime, China). Besides, the Total Superoxide Dismutase Assay Kit with WST-8 method was applied to test the activity of superoxide dismutase (SOD).

Apoptosis was analyzed by flow cytometry using Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) detection kit (Beyotime, China). Briefly, the third-passage ASCs were seeded in 6-well plates in GM at a density of 5 × 10⁴ cells/ml. After 24 h culture, ASCs were pretreated with 100 ng/ml irisin for 2 h and then treated with or without 40 μg/ml AGEs for another 48 h in the presence of irisin. Then, cells were collected and incubated with a mixture containing Annexin V-FITC and PI for 20 min in the dark and detected on a BD Influx flow cytometer (BD Biosciences, San Jose, CA). The total apoptosis rate of ASCs was calculated as the sum of rates of cells observed in the lower-right quadrant (early-phase apoptotic cells) and the upper-right quadrant (late-phase apoptotic/necrotic cells).

Samples for flow cytometry were stained with SYBR Green I (1:10000), incubated for 20 min, and run on a BD Influx flow cytometer (BD, Franklin Lakes, NJ, USA) (488 nm laser; emission 530 nm, 40 nm band pass) on a linear scale with at least 10,000 T. pseudonana cells analyzed per sample [10 (link)]. T. pseudonana cells were gated using the FCSplankton package in R (https://github.com/fribalet/FCSplankton). SYBR Green I-stained cells were manually gated on forward scatter and 530/40 nm emission using the set_gating_params() function. Forward scatter distributions, as a proxy for cell size, were normalized to 1 μm beads (Molecular Probes, Eugene, OR, USA) that were added to each sample as an internal standard. Contrary to control cultures, the G1, S, and G2 populations of the filtrate-treated cultures did not follow a Gaussian distribution, precluding quantitative cell cycle decomposition of these samples. To compare the distributions of the SYBR-stained control and filtrate-treated cells, the distributions were aligned to the mode of the G1 peak. Cell cycle stage boundaries were drawn based on the signal of the control cultures, allowing for qualitative comparison of the G1, S, and G2 population distributions between control and filtrate treated cultures.