Fitc conjugated anti ly6g

Cells at D3 CMP stage were treated with 20 nM RIST4721 (or DMSO) and differentiated into mature neutrophils as described above while replenishing the drug and DMSO during culture expansion or media changes. Cell surface marker analysis using fluorescent immunolabeling and imaging flow cytometry was then performed as previously described [42 (link)]. Briefly, the cells were harvested at 1 × 10⁶ per sample through centrifugation at 250× g for 5 min. The pellets were then washed in PBS and centrifuged again. The cells were resuspended in PBS with 2% FBS at 100 μL per sample, and Fc Block (Purified Rat Anti-Mouse CD16/CD32; BD Biosciences) was added into each sample at 50 μg/mL followed by incubation on ice for 15 min. The cells were then stained with PE-conjugated anti-Cd11b, FITC-conjugated anti-Ly6G, or corresponding isotypes (4 μg/mL for each antibody; BD Biosciences) for 45 min on ice while protected from light. Finally, the samples were washed in 400 μL of PBS with 2% FBS, centrifuged at 1400× g for 5 min and resuspended in 35 μL of fresh PBS with 2% FBS for processing with FlowSight imaging flow cytometer (Luminex, Austin, TX, USA).

Flow cytometry was used to analyze the distribution of peripheral blood leukocytes and T cell subpopulation in whole blood. Each blood sample was divided into 100 μL in each aliquot, and these were then incubated with the different antibodies as described below. The antibodies used to detect leukocytes were as follows: PerCP-conjugated anti-CD45 (Biolegend, San Diego, CA, USA) for leukocytes, PE-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA) for monocytes/macrophages and FITC-conjugated anti-Ly6G (BD Biosciences, San Jose, CA, USA) for neutrophils. For analysis of the T cell subpopulation, blood was incubated with PerCP-conjugated anti-CD45 (Biolegend), APC-conjugated anti-CD3ε (eBioscience), FITC-conjugated anti-CD4 (eBioscience) and Pacific blue-conjugated anti-CD8 (Biolegend) antibodies. The concentrations of antibodies were compliant with those recommended by the manufacturer. After 30 min of incubation at 4 °C in the dark, the lysed red blood cells were first suspended in staining buffer and then analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated for analysis, with the results presented as a percentage of specific CD-marker-expressing cells in blood leukocytes.

Zymosan A (ZyA; Alfa Aesar), DMSO (Tocris Bioscience), Rapamycin (Adipogen), 6-Diazo-5-oxo-L-norleucine (DON; Sigma-Aldrich), Torin1 (Tocris Bioscience), compound 968 (C968; Calbiochem) were purchased. In vitro experiments used the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS; MP Biomedicals), 1% penicillin-streptomycin (Lonza Walkersville), and recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech). For flow cytometry analysis of myeloid cells, we used FITC-conjugated anti-Gr1 (BD PharMingen), FITC-conjugated anti-Ly6G (BD PharMingen), PerCP-conjugated anti-CD11b (BioLegend), APC-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD11c (eBioscience), and PE-conjugated anti-F4/80 (eBioscience). For the flow cytometry analysis of lymphocytes, we used FITC-conjugated anti-CD4 (eBioscience), APC-conjugated anti-Rorγt (eBioscience), PE-conjugated anti-Foxp3 (eBioscience), and 7-AAD Viability Staining Solution (eBioscience). For the Western blotting analysis, anti-phospho-p70 S6 kinase (Thr389) (Cell Signaling, #97596 S), anti-p70 S6 kinase (Cell Signaling, #9202), anti-phospho-Akt (Ser473) (Cell Signaling, #4060), anti-Akt (Cell Signaling, #2920), and anti-β-actin antibodies (Sigma-Aldrich) were purchased.