Rat anti mouse igm fitc clone 2 41

The following antibodies were used to label the different PBMC subpopulations: anti-ovine CD4 (clone 44.38), CD8 (clone 38.65), and WC1 (clone 19.19), anti-human CD14 (clone TÜK4) and CD16 (clone KD1) (all from Bio-Rad), and anti-bovine B cell marker (clone BAQ44A) (Kingfisher Biotech). PBMC flow cytometry was performed as described in (1 (link)). Briefly, PBMCs were washed twice in staining buffer (PBS + 2% FBS + 0.02% sodium azide) and stained for 20 min on ice with the appropriate fluorochrome-conjugated surface antibodies. In the case of the unconjugated anti-bovine B-cell marker, a rat anti-mouse IgM FITC (clone II/41, BD Biosciences) was used as secondary antibody. Samples were run on a FACSCalibur (Becton Dickinson) flow cytometer. Dead cells were excluded from the analysis by 7-AAD staining (BD Biosciences) and gating was performed using the appropriate isotype controls. Analysis was performed using FlowJo software (TreeStar Inc.).

The following antibodies were used in the present work to characterize PBMC cell populations: anti-human CD16 (clone KD1, Biorad), anti-CD3 (clone CD3-12, Biorad), anti-bovine B cell marker (clone BAQ44A, Kingfisher Biotech), anti-human CD14 (clone TÜK4, Biorad). Anti-FLAG (F7425, Sigma) and anti-HA (clone 6E2, Cell Signaling) antibodies were used to detect protein expression in transfected cells. Anti-GAPDH antibody was used as sample loading control for western blot. Anti-ovine MHC-I antibody (clone 41.17, Biorad) was used as positive control to label cells in antibody dependent cell cytotoxicity assays. Anti-PPRV-N monoclonal antibody was used 1:100 for PPRV detection (Dr. Libeau, CIRAD, Montpellier, France) in flow cytometry experiments. Anti-BTV-VP7 monoclonal antibody (VMRD; CJ-F-BTV-MAB-10 ML) was used for BTV detection. Rat anti-Mouse IgM-FITC (Clone II/41, BD biosciences), anti-mouse IgG-Alexa 488 or 647 (Thermofisher), anti-sheep-IgG Alexa 488 (Thermofisher) were used as secondary antibodies for flow cytometry, and immunofluorescence. Anti-Mouse IgG or anti-Rabbit IgG HRP-conjugated secondary antibodies (GE Healthcare) were used for western blots.