Anti mouse cd3 bv421

Bone marrow was flushed from 6–8 week old mice using 1X PBS with 2% FBS. Red blood cells were lysed using 1X RBC lysis buffer (Biolegend). Cells were washed twice in 1X PBS with 0.5% BSA, then blocked using rat IgG (TruStain fcX, Biolegend) for 10 minutes on ice. Cells (1 × 10⁶ cells in 100 μl) were then detected with antibodies at 0.2 μg per million cells for 30 minutes on ice protected from light. The antibodies used for flow cytometry to detect osteoclast precursors were the following: anti-mouse CD3-BV421 (BD Biosciences #564008), anti-mouse CD45R-Alexa Fluor488 (BD Biosciences #557669), anti-mouse CD11b-APC (BD Biosciences #553312), anti-mouse CD115-PE (BD Biosciences #565249). Flow cytometry was conducted with a LSR II (BD Biosciences) and data were analyzed using FlowJo software.

Intestinal epithelial cell isolation was performed as previously described 39. Briefly, the colon was removed and placed in a tissue culture medium (RPMI 1640, with 2% fetal calf serum). Then, the colon was cut into 5-mm pieces followed by extensively rinsed with ice-cold PBS containing 2% fetal calf serum. The rinsed pieces were then incubated in Ca2⁺- and Mg2⁺-free PBS containing 5 mM EDTA, 2 mM DTT and 10% fetal calf serum for 20 min at 37 °C with continuous brisk stirring. Then, the supernatant was collected and filtered through both 70 and 30 μm MACS SmartStrainers (Miltenyi Biotec) to remove debris and pellets. Intestinal epithelial cells and IELs obtained by centrifugation of collected supernatant can be used directly for flow staining.
After centrifugation, the IELs were purified by 40%/70% Percoll (GE Healthcare Bio-sciences) from Intestinal epithelial cells. Then, the CD3e MicroBead Kit (Miltenyi Biotec) was used to select CD3⁺ intraepithelial lymphocytes according to the manufacturer’s instructions. The purity of the IELs was detected by flow cytometric analysis. The IELs were stained with Bv421 anti-mouse CD3 (BD Biosciences) according to the manufacturer's protocol. The acquisition and analysis were performed using Beckman Coulter Gallios Flow Cytometer (Beckman Coulter).