Pe conjugated anti ly6g

For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).

At day 45 p.t.i. mice with residual primary lymphoma from CHOP×2 group (i.e. animals with partial response to chemotherapy) as well as PBS control, were sacrificed and tumors were removed and prepared as a single-cell suspension. Cells were immunostained at 4 °C in the dark for 30 min with the following panel of antibodies: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, PE-conjugated anti-Ly6G, (all reagents from BD Pharmingen, San Diego, CA). Optimal antibody concentration was previously defined by titration. For intracellular FoxP3 staining, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) according to the manufacturer’s protocols. Cells were washed twice with permeabilization buffer and then incubated with anti-FoxP3 at 4 °C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used.

At day 45 p.t.i., five mice from PBS and LVR01x3 groups and five mice with residual lymphoma (partial response) from CHOPx2 and CHOPx2 + LVR01x3 were sacrificed and tumors were removed and prepared to obtain a single-cell suspension. 1 × 10⁵ cells per tumor were immunostained at 4°C in the dark for 30 min with the following antibodies panel: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, and PE-conjugated anti-Ly6G (all reagents from BD Pharmingen, San Diego, CA, USA). The optimal antibody concentration was defined by titration. For Treg cells analysis, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) and then washed twice with permeabilization buffer and incubated with anti-FoxP3 at 4°C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II Cytometer (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used, respectively.

For immunoblot analysis, S087 rabbit immune serum was used to detect both Rab27a and Rab27b. Slp1 and Munc13-4 rabbit immune serum used for immunoblot were kindly provided by Dr. Sergo D. Catz (The Scripps Research Institute, La Jolla, CA, USA) and were described previously [17 (link),37 (link)]. Immunoblots were performed as described previously [14 (link)]. For flow cytometry using a FACScalibur cytometer, phycoerythrin (PE)-conjugated anti-Ly6G (BD Biosciences) and allophycocyanin (APC)-conjugated CXCR2 (R&D Systems) antibodies were used at 1:100 dilution. For surface antigen staining, 2 × 10⁵ bone marrow derived neutrophil (BMDN) cells were washed twice and resuspended in rat anti-FcγRII/RIII antibody (BD biosciences) for 10 minutes, then incubated with fluorophore-conjugated antibodies for 30 minutes on ice.