CD3e Monoclonal Antibody (145-2C11), Biotin, eBioscience™

Protocol tips

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	CD3 (clone 145-2C11), corresponding isotype controls, and secondary reagents (efluor450-, APC-efluor780–, and PE-Cy7–conjugated streptavidin) were purchased from eBioscience.

Protocol tips
CD3 (clone 145-2C11), corresponding isotype controls, and secondary reagents (efluor450-, APC-efluor780–, and PE-Cy7–conjugated streptavidin) were purchased from eBioscience.

Publication protocol

Fluorochrome-conjugated or biotinylated mAbs specific to mouse Gr-1 (Ly6G/C; clone RB6-8C5), CD115 (clone AFS98), Siglec-F (clone E50-2440), CD11b (clone M1/70), CD11c (clone N418), I-A/I-E clone (clone M5/114.15.2), CD45 (clone 30-F11), Sca-1 (clone D7), Flk2 (clone A2F10), CD117 (clone 2B8), CD3 (clone 145-2C11), B220 (clone RA3-6B2), Ter119 (clone TER-119), CD51 (clone RMV-7), and CD31 (clone MEC13.3), corresponding isotype controls, and secondary reagents (efluor450-, APC-efluor780–, and PE-Cy7–conjugated streptavidin) were purchased from eBioscience. Anti-F4/80 (clone CI:A3.1), CD68 (clone FA-11), and CD169 (clone 3D6.112) were purchased from AbD Serotec. CD68 was stained extracellularly and subsequently intracellularly with the Cytofix/Cytoperm kit (BD) according to the manufacturer’s protocol. Multiparameter analyses of stained cell suspensions were performed on an LSRII (BD) and analyzed with FlowJo software (Tree Star, Inc.). DAPI− single cells were evaluated for all analyses except for intracellular stains. To purify mononuclear phagocyte populations, BM was sorted with an InFlux cell sorter (BD) to achieve >97% purity. Sorted mononuclear phagocytes were cytospun with Cytospin 3 (Thermo Fisher Scientific) and stained with Hema 3 manual staining system (Thermo Fisher Scientific). To isolate Nestin+ and Nestin− cells from the BM for Q-PCR, RBC-lysed BM cells were digested with collagenase, trypsin, and DNase, as previously described (Méndez-Ferrer et al., 2010b). Endothelial cells and osteoblasts were isolated similar to previous studies (Semerad et al., 2005; Winkler et al., 2010). In brief, tibias, femurs, and humeri of mice were flushed thoroughly of BM cells, chopped with a scalpel, and washed three times through a 5-ml polystyrene tube with blue-top cell strainer (BD) to further remove residual BM cells. The bone fragments were then digested at 37°C with Type IA collagenase (Sigma-Aldrich) for 40 min while spinning. RBC-lysed pellet was then stained for sorting. Cells were sorted by Moflo Cell sorter (Dako) at the Flow Cytometry Core Facility at Mount Sinai School of Medicine or Aria Cell sorter (BD) at the Flow Cytometry Core Facility at Albert Einstein College of Medicine.

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Manufacturer protocol

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