Anti annexin 5

L1210 cells (3 × 10⁵) were incubated with mSIRPα^ext (10 µg/mL) for 30 min on ice in dark. After washing with PBS, cells were stained with FITC-conjugated anti-S tag antibody (Sigma), followed by FACS analysis using a Calibur™ (BD Immunocytometry Systems, San Jose, CA, USA). BMDMs (3 × 10⁵) were cultured in the presence of PBS (M^PBS) or LPS + IFNγ (M^LPS) for 24 h, and then incubated with mSIRPα^ext (10 µg/mL) at 37°C for 2 h. After washing, cells were stained with anti-Ki67 (Sigma) or anti-Annexin V (BD), followed by FACS analysis. Data were analyzed using the Flowjo™ software.

For apoptosis staining, 1 × 10⁶ cells of each sample were stained with anti‐Annexin‐V (BD Biosciences, San Jose, CA, USA) and 7‐AAD (eBioscience, San Diego, CA, USA). For staining of CCR5 and CXCR4, 1 × 10⁶ cells per sample were stained with anti‐human CCR5 PE‐conjugated antibody (R & D, Minneapolis, MN, USA) or PE anti‐human CD184 (BD Pharmingen, San Jose, CA, USA). Intracellular p‐Akt, p‐Stat5, and Bcl‐2 staining was performed with 1 × 10⁶ cells, which had been starved overnight, according to the manufacturer’s protocol. Briefly, cells were fixed with Cytofix Buffer (BD Biosciences) at 37°C for 15 min. Afterward, cells were permeabilized for 30 min on ice using Perm Buffer III (BD Biosciences, San Jose, USA) followed by two washing steps and were stained with Alexa 647 anti‐p‐Akt (pS473) (BD Biosciences), Alexa 647 anti‐p‐Stat5 (pY694) (BD, Biosciences) for 1 h or with PE anti‐Bcl‐2 (BD Biosciences) for 30 min. All samples were analyzed on a Fortessa cytometer (BD Biosciences), and data analysis was performed using flowjo V10 software (FlowJo LLC, Ashland, OR, USA).

Mice were injected with 2 mg of BrdU 60 min before harvest; incorporation levels were assessed using kit (Cat#552598; BD Biosciences). Apoptosis rates were determined by flow with anti-annexinV (BD Biosciences).