Fitc anti gr 1

Spleen and inguinal lymph node (LN) cells were harvested from immunized mice at the time of sacrifice. Joint tissues were minced and treated with collagenase (0.25 mg/ml), and joint cell populations were examined for surface markers using antibodies anti-CD4-AF700, anti-CD8a-PE-Cy7, anti-CD19-PE, anti-CD11b-Pacific Blue, anti-CD11c-APC-Cy7, anti-F4/80-APC, and anti-GR-1-FITC (BD Pharmingen, San Jose, CA, USA). For intracellular cytokine staining, cells were stimulated with Phorbol 12-myristate 13-acetate (25 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) and treated with GolgiPlug (BD Pharmingen) for 4 hours. After cell surface staining with anti-CD3e-PE-Cy7 and anti-CD4-APC-Cy7, cells were permeabilized using the Cytofix/Cytoperm Plus kit (BD Pharmingen) and stained with anti-IFNγ-APC and anti-IL-17A-FITC. A BD LSRII cytometer was used for cytometry and data were analyzed using BD FACS Diva Software.

Staining for cellular and nuclear morphology was performed on cytospins from cultures of progenitors or differentiated neutrophils by incubating with Giemsa solution (Merck, Darmstadt, Germany) after methanol fixation. Analysis by brightfield microscopy was performed using a Keyence BZ9000 microscope at a magnification of x40 (Keyence, Neu-Isenburg, Germany).
Expression of cell surface markers was measured by staining cells with anti-Gr-1-FITC (BD Biosciences, San Jose, CA, USA), anti-CD11b-APC (eBioscience, San Diego, CA, USA), anti-c-kit-APC (eBioscience) or anti-CXCR2-APC (Biolegend, San Diego, CA, USA) followed by flow cytometry analysis on a FACS Calibur (BD Biosciences, Heidelberg, Germany).

After being harvested and weighed, tumor tissues were cut into small pieces and mixed with IV collagenase for 2 h. Tissues were homogenized and filtered through a 70 μm filter to remove remaining undigested tissue. The single cell suspension was centrifuged at 500 g for 5 min, incubated with 2 μl extracellular antibody for 30 min, and then fixed with 4% paraformaldehyde for 20 min. Finally, the suspension was incubated with intracellular antibody for another 30 min before being tested by flow cytometry (NovoCyte, ACEA Biosciences, San Diego, USA). APC-Anti-CD4 (#553051), PE-Cy7-anti-CD25(#552880), PE-anti-FOXP3(#563101), PE-Cy7-anti-CD11b (#552850) and FITC-anti-Gr-1(#553126) were purchased from BD pharmingen™.

The anti‐α‐tubulin antibody, aphidicolin, and nocodazole were purchased from Sigma‐Aldrich. The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz. Antibodies against ATM, Flag, mouse cGAS, human cGAS, STING, MRE11, PARP1, H2A, H2A.X, γ‐H2A.X p‐IRF3, and IRF3 were from Cell Signaling Technology; Alexa 488‐anti‐Sca‐1 was from Invitrogen and PECY7‐anti‐cKit; V450‐Ly6G and FITC‐anti‐GR1 were from BD Pharmingen; 2′,3′‐cGAMP and immunostimulatory DNA (ISD) were from InvivoGen; and ATP was from New England Biology, while GTP, Rad51, and Lamin B1 antibody were from Abcam.

The following conjugated anti-murine antibodies were used for flow cytometry: FITC-anti-CD45.1 (A20, #11-0453), FITC-anti-CD9 (MZ3, #11-0091), PE-Cy7-anti-CD45.2 (104, #12-0454), PE-anti-CD127 (A7R34, #12–1,271), efluor450-anti-CD19 (eBio1D3, #48-0193), efluor-anti-CD45.2 (104, #48-0454), PECy7-anti-CD117 (2B8, #25–1,171), efluor-anti-CD34 (RAM34, #50-0341; eBioscience); APC-Cy7–anti-B220 (RA3-6B2, #103224), APC-anti-CD22 (OX-97, #126110), PE-Cy7-anti-CD93 (AA4.1, 136506; Biolegend); PE-anti-CD22 (Cy34.1, #553384), APC-anti-CD11b (M1/70, #553312), PE-anti-BP1 (BP-1, #553735), FITC-anti-GR1 (RB6-8C5, #553127; BD Biosciences). CAR detection was performed using protein-L with PE-Streptavidin (BD Biosciences). The following conjugated anti-human antibodies were used: efluor45-anti-CD19 (HIB19, #48-0199), FITC-anti-CD19 (HIB19, #11-0199)), efluor-anti-CD34 (4H11, #48-0349), PE-Cy7-anti-CD11b (ICRF44, #25-0118), FITC-anti-CD14 (61D3, #11-0149), APC-anti-CD117 (104D2, #17–1,178), PE-anti-CD33 (WM-53, #12-0338), PerCP-Cy5.5-anti-CD45 (HI30, #45-0459; eBioscience); PE/Cy7-anti-CD10 (H110a, #312214, Biolegend); PE-anti-CD22 (S-HCL1, #347577) and PE-anti-HLA-DR (G46-6 #556644; BD Biosciences). Samples were analysed on a BD LSR-Fortessa (BD Biosciences), data collected using FACS Diva software and analysed using FLowJo version 9.6.4 (Treestar).

For analysis of multilineage blood cells, FITC‐Anti‐GR‐1 (BD), FITC‐Anti‐CD45R/B220 and FITC‐Anti‐CD11b antibodies were used for the test of the peripheral blood. And the proportion of positive marked cells was measured on a flow cytometer (BD Accuri™ C6 Plus).