Online supplementary text

Supplementary Table S1: primer sequences used for real-time PCR

Gene / Primer sequence
GAPDH / FW 5’-GGC AAA TTC AAC GGC ACA-3’
RV 5’-GTT AGT GGG GTC TCG CTC CTG-3’
FcRI / FW 5’-ACA CAA TGG TTT ATC AAC GGA ACA-3’
RV 5’-TGG CCT CTG GGA TGC TAT AAC T-3’
FcRIIB / FW 5’-GAC AGC CGT GCT AAA TCT TGC T-3’
RV 5’-GTG TCA CCG TGT CTT CCT TGA G-3’
FcRIII / FW 5’-GAC AGG CAG AGT GCA GCT CTT-3’
RV 5’-TGT CTT CCT TGA GCA CCT GGA T-3’
FcRIV / FW 5’-CCA GAG TTA AGG ACA GTG GAA TGT AC-3’
RV 5’-GCA ATA GCC AGC CCA TAT GG-3’
CTR / FW 5’-CGT TCT TTA TTA CCT GGC TCT TGT G-3’
RV 5’-TCT GGC AGC TAA GGT TCT TGA AA-3’
NFATc1 / FW 5’-ATG CGA GCC ATC ATC GA-3’
RV 5’-TGG GAT GTG AAC TCG GAA GAC-3’
DC-STAMP / FW 5’-TGT ATC GGC TCA TCT CCT CCA T-3’
RV 5’-GAC TCC TTG GGT TCC TTG CTT-3’
TRACP / FW 5’-GAC AAG AGG TTC CAG GAG ACC-3’
RV 5’-GGG CTG GGG AAG TTC CAG-3’
Cathepsin K / FW 5’-GAA GCA GTA TAA CAG CAA GGT GGA T-3’
RV 5’-TGT CTC CCA AGT GGT TCA TGG-3’
F4/80 / FW 5’-ACT GTG GAA AGC ACC ATG TTA G-3’
RV 5’-GCT GCC AAG TTA ATG GAC TCA-3’

Supplementary Figure S1:

Effect of IC stimulation on osteoclast differentiation and function using BSA:anti-BSA ICs. Unfractionated bone marrow cells were either differentiated towards osteoclasts in the presence of ICs for 6 days. IC were formed in a ratio of 1 BSA: 5 anti-BSA corresponding with a final concentration BSA of 500 g/ml and anti-BSA of 200 g/ml. Additionally, BSA (500 g/ml) and anti-BSA (200 g/ml) were used as negative controls. (A) Formation of TRACP+ multinucleated cells (MNCs). Values are mean ± SEM (n = 6 mice per group). Significance was tested by one-way ANOVA (** = P < 0.01). (B) Bone resorption levels of IC-stimulated osteoclasts and controls. Data are expressed as mean ± SEM (n = 6 mice). Significance was tested by Kruskal-Wallis test (** = P < 0.01).

Supplementary Figure S2:

To determine FcγR expression levels on bone marrow progenitors, 6 bone marrow subsets were discriminated using ER-MP12 and ER-MP20 rat monoclonal antibodies recognizing CD31 and Ly-6C, respectively. For simultaneous analysis of FcγR expression levels on these bone marrow subsets, 1x106 freshly isolated bone marrow cells per sample were first incubated with biotinylated ER-MP20. Subsequently, cells were incubated with FITC-conjugated ER-MP12, streptavidin-PE, and Alexa647- or APC-conjugated monoclonal antibodies for detection of FcγRs (Alexa647-FcγRI, Alexa647-FcγRIV or APC-FcγRII/III). A) Gates indicate the six phenotypically distinct subpopulations: 1) early blasts, 2) lymphoid cells, 3) erythroid cells, 4) myeloid blasts, 5) granulocytes, and 6) monocytes. B) Solid black lines in the histograms indicate FcγR expression on osteoclast precursor subsets (early blasts, myeloid blasts, and monocytes). Grey filled loops represent isotype controls.