Goat anti chicken igm

PBMCs were isolated by Ficoll (Sigma Aldrich) density gradient centrifugation from EDTA‐treated blood or splenocyte cell suspensions. The following mouse‐anti‐chicken antibodies were used: Bu‐1 (AV20, conjugated or unconjugated), lambda‐PE (L‐1), TCR gamma/delta (TCR1), TCRαβ/Vβ1 (TCR2), TCR αβ/Vβ2 (TCR3), monocytes/macrophage marker (KUL01), Cμ‐CH1 (M1, conjugated or unconjugated), CD4 (CT4), and CD8 (CT8) (all from Southern Biotech), ms‐anti‐chicken‐Cμ (BK) made by Bernd Kaspers (binds a part of the constant region of Cμ other than CH1), or goat‐anti‐chicken‐IgM (Bethyl Laboratories). Secondary antibodies were: goat‐anti‐mouse‐IgG‐Alexa‐647 (Rockland), goat‐anti‐mouse‐IgG‐FITC (Thermo Fisher Scientific), or donkey‐anti‐goat‐IgG‐Alexa‐647 (Rockland). Fluorescence was measured using an Attune^TM Flow Cytometer (Thermo Fisher Scientific).

SYK-deficient DT40 cells were electroporated with 30 µg of the indicated eDHFR-linked protein expression plasmid or mock plasmid and 10 µg NFAT- or NFκB-luciferase plasmid and then allowed to recover for 24–48 h. The cells were collected and resuspended in medium lacking chicken serum at a density of 1 × 10⁶ cells/mL. Cells were treated with either 1 µM ionomycin plus 50 ng/mL PMA, 0.5% dimethylsulfoxide (DMSO), 0.5 µg/mL goat anti-chicken IgM (Bethyl Laboratories, Montgomery, TX, USA), anti-IgM plus 2 µM Latrunculin B (LatB, AdipoGen, San Diego, CA, USA), or anti-IgM plus the indicated concentration of TMP dimer for 6 h at 37 °C. Luciferase activity was measured using a Promega Luciferase Assay System kit per manufacturer instructions. All measured values were normalized to the PMA/ionomycin internal control. All experiments consisted of three technical replicates and were repeated 3–4 times.

To measure total plasma IgM and IgY EIA plates (Corning) were coated with 2 μg/mL goat‐anti‐chicken‐IgM (Bethyl Laboratories) or 0.8 μg/mL goat‐anti‐chicken‐IgY (Sigma Aldrich) overnight at 4°C. Plates were blocked with PBS/3% skim milk and incubated with chicken plasma. Bound IgM or IgY was detected with goat‐anti‐chicken‐IgM‐HRP (Bethyl Laboratories) or goat‐anti‐chicken‐IgY‐HRP (Sigma Aldrich) and developed with TMB substrate (Thermo Fisher Scientific). OD was measured at 450 nm. To measure antigen‐specific IgM and IgY, plates were coated with 10 μg/mL KLH or BSA. For detection of the Cμ‐CH1 domain plates were coated with 2 μg/mL ms‐anti‐chicken‐ Cμ‐CH1 (M1) (Southern Biotech). All subsequent steps were performed as for detection of total plasma IgM and IgY.