Cd11b pe

Whole bone marrow was isolated and stained on ice with various antibody cocktails to identify each progenitor compartment. PE-CD11b and FITC-Ly6G (BD Science, San Jose, CA, USA) were used to detect the neutrophil populations, while PE-CD184 (BD Science, San Jose, CA, USA) was used to detect the CXCR4⁺ cell populations. Biotin-CD4, Biotin-CD8a, Biotin Mouse Lineage Panel, PE-CD135, V450-CD127, APC-CD117, PE-Cy7 Streptavidin (BD Science, San Jose, CA, USA), PE-Cy5.5 Sca1 (Abcam, Cambridge, UK), and AF700-CD16/32 (eBioscience, San Diego, CA, USA) were used to detect the HSCs and their differentiated cell populations. All the labeled cells were evaluated using an Aria II flow cytometer (BD Science, San Jose, CA, USA) or FC500 flow cytometer (Beckman, Brea, CA, USA), and the data were processed using FlowJo software (BD Science, San Jose, CA, USA). For detailed procedure, see Supplementary Figure 2.

To deplete MΦs in the draining lymph nodes as well as in the blood system and spleen (20 (link)), 200 μL of liposomes carrying 12 μg of clodronate (Lp/Clodronate) were injected by intraperitoneal route (i.p.) every 3 days, starting 6 days before the first immunization. One group received 200 μL of liposomes without clodronate as control. To check depletion of MΦs, cell suspensions from the peritoneal cavity (PerC) were pre-harvested and incubated with an anti-CD16/CD32 mAb (BD Biosciences Pharmingen, San Diego, CA, United States) to block Fcγ II/III receptors before staining with fluorochrome-conjugated antibodies. Cells were stained with the following combination of goat anti-mouse antibodies: FITC–CD19, PE-CD11b, PE-Cy5-F4/80, and PE Cy7-CD11c (BD Biosciences Pharmingen), using standard protocols. Cells were acquired with a Gallios flow cytometer (Beckman Coulter, Miami, FL, United States). The analysis was performed using FlowJo 7.2.2 software (Tree Star Ashland, OR, United States). The total number of MΦs was estimated by total cell number in the PerC counted in a Neubauer chamber.

THP-1 cells were differentiated using 200 nM 12-myristate 13-acetate (PMA, Sigma–Aldrich) as previously described^{25 (link)}. FACS was used for the detection of CD14, CD11b, F4/80, and CD11c. Briefly, macrophages were harvested and stained with FITC-CD14, PE-CD11b, PE-F4/80, and FITC-CD11c antibodies (BD Biosciences), followed by four-color FACS analysis with FACSCailbur (BD Biosciences). All data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).

For wild type (WT), DβYTL and Dβ2ko mice, single cell suspensions were prepared from the thymus of two mice each. Red blood cells were removed with RBC lysing solution (1 mM KHCO₃, 0.15 M NH₄Cl, and 0.1 mM Na₂EDTA). Cells were washed twice and resuspended in a master-mix of staining buffer containing optimal concentrations of monoclonal antibody reagents. Samples were sorted with a FACS Aria (Becton Dickinson). Double negative thymic cells were stained with PE-Cy7-CD25 (BD Cat #552880), APC-CD44 (BD Cat #559250), biotinylated-CD28 (BD Cat #553296) (developed secondarily with streptavidin), and a lineage stain [PE-CD3 (BD Cat #555275), monoclonal PE-CD4 (BD Cat #553049), PE-CD8α (BD Cat #553033), PE-B220 (BD Cat #561878), PE-CD11b (BD Cat #553311), PE-NK1.1 (BD Cat#553165)] to remove mature T, B, and NK cells. The cells were stained with propidium iodide (PI) to identify/sort live cells. DN2 cells were defined as CD44⁺ and CD25⁺ (Supplementary Figure S2).

Phorbol-12-Myristate-13-acetate (PMA, 100 ng/mL; Catalog No. P1585; Sigma-Aldrich; Merck KGAA) was taken to differentiate THP-1 cells (24 (link)). The differentiated THP-1 cells were dyed with FITC-CD14, PE-CD11b, PE-F4/80, and FITC-CD11c antibodies (BD Biosciences) and incubated in darkness at indoor temperature for 15 minutes. Eventually, flow cytometry (BD Biosciences) analyzed the stained cells with the help of FACS Calibur Flow Cytometer (BD Biosciences, Sanjose, CA, USA). The whole process was conducted in triplicate.