Goat anti human igg a647

Systemic IgG and secretory IgA binding to microbiota was assessed by bacterial flow cytometry as previously described.^{21 (link)} Briefly, thawed microbiota or bacterial strains (10⁷ bacteria/conditions) were fixed in a solution of 4% paraformaldehyde and stained with Cell Proliferation Dye eFluor 450 (eBioscience). After washing with 1xPBS (10 minutes, 4,000g, 4°C), cells were suspended in 1xPBS, 2% bovine serum albumin (Sigma), and 0.02% sodium azide (Sigma) and incubated in a 96-V bottom well plate with a 10 μg/mL IgG solution (from either human serum or pooled human IgG Hizentra–CSL Behring France or human anti-tumor necrosis factor (TNF) Remicade^–MSD France) per condition. All buffers were passed through sterile 0.22-μm filters before use. After washing, secondary conjugated antibodies (goat anti-human IgA-FITC and goat anti-human IgG-A647) or isotype controls (both from Jackson ImmunoResearch Laboratories, West Grove) were added for 20 minutes at 4°C. Then, bacteria were suspended in sterile PBS. Thirty thousand bacterial events were acquired on a FACSCanto II flow cytometer (Becton Dickinson). Analysis was performed with FlowJo software (Treestar). Frequencies of Ig-bound microbiota were expressed as percentages, median, minimum, and maximum values throughout the article. Medians of fluorescence were used to measure IgG-binding levels for pure bacterial strains.