The rat AG-014699 purchase myeloid cell line RMW [5] and BWN3G [27] were generated in our laboratory. 293T cells were obtained from ATCC. Resident peritoneal macrophages were collected by peritoneal lavage, left to adhere to plastic dishes for 2 h, and washed. Remaining adherent cells were cultured overnight in either M-CSF (20 ng/mL), IL-4 (20 ng/mL), or IFN-γ (20 ng/mL) plus LPS (10 ng/mL) (cytokines from PeproTech and LPS from InvivoGen). Cells were analyzed by flow cytometry the next day. Expression constructs consisting of the full-length

open reading frames of rat Mincle, DCIR-1, or KLRH1 followed by a C-terminal FLAG epitope tag; rat FcεRI-γ with an N-terminal HA tag; or rat MCL without tag were generated. 293T cells were transiently transfected using polyethylenimine “Max” (m.w. 25 000, Polysciences) [28]. A rat Mincle-Fcγ2b Fc fusion protein was used to immunize female BALB/c mice by i.p. injections. Hybridomas were generated using standard techniques. One clone of IgG1 isotype, WEN43,

was selected and shown by flow cytometry to react specifically with cells transfected with rat Mincle, and not other APLEC-encoded receptors (Fig. 1). WEN42 (anti-MCL), WEN43 (anti-Mincle), and OX-42 (anti-CD11b/c) were produced in-house; STOK9 (anti-KLRH1) was a gift from Bent Rolstad; commercial antibodies were OX-41 (anti-CD172a/SIRP-α, Accurate Chemical & Scientific Co.), OX-22 (anti-CD45RC, BioLegend), and OX6 (anti-MHC class II, AbD Serotec). Data were acquired using a FACSCanto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). Transfected 293T cells were lysed in 1% digitonin (Calbiochem) lysis selleck compound buffer. Lysates were immunoprecipitated with Protein G Dynabeads (Invitrogen), separated by SDS-PAGE, and detected by ECL as detailed previously [5]. For immunoprecipitation, mAbs used were: anti-MCL (WEN42), see more isotype control (W6/32, IgG2a), or anti-FLAG (M2, Sigma-Aldrich). Immunoblotting: anti-FLAG-biotin (M2, Sigma-Aldrich), anti-MCL-biotin (WEN42), or rabbit anti-FcεRI-γ (Upstate Biotechnology) were used as primary antibodies, followed by streptavidin-HRP or HRP-conjugated anti-rabbit IgG

(Jackson ImmunoReseach Laboratories). 293T cells were incubated with Ab-coated yellow-green fluorescent 1-μm microspheres, counterstained with streptavidin-DyLight594, and analyzed by imaging flow cytometry on an ImageStream X (Amnis) as described previously [5]. For blocking, a cocktail of three mAbs specific for rat MCL was used. To compare efficiency of phagocytosis, data are expressed as phagocytic ratio: (fraction of bead-binding cells with internalized beads)/(fraction of bead-binding cells with no internalized beads). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison posttest (GraphPad InStat). We would like to thank Wendi Jensen for her technical assistance. This work was supported by grants from the University of Oslo (to M.R.D.) and the Norwegian Research Council, Norway (to M.R.D., S.F.