Pe conjugated anti f4 80

Single cell suspensions (0.5 x 10⁶ cells) were incubated for 15 min with Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), washed with PBS supplemented with 2% FCS and then stained for 30 min with the indicated conjugated antibodies. Cells were washed twice and fixed with 0.37% formaldehyde in PBS. FITC-conjugated anti-CD19, PE-conjugated anti-CD3, APC-conjugated anti-CD4, BV410-conjugated anti-CD8, APC-conjugated anti-CD11b, BV711-conjugated anti-CD11c, FITC-conjugated anti-Gr-1 and PE-conjugated anti-F4/80, were purchased from eBioscience (San Diego, CA, USA). Cells were analysed in a FACS Fortessa flow cytometer and data were processed with the FlowJo software (Becton Dickinson Labware, Franklin Lakes, NJ, USA).

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.

Groups of 8-wk-old male BALB/c mice were intraperitoneally injected with 200 ul PBS containing with and without LPS (1 mg/Kg of weight; E. coli O55:B5; Sigma). After 16 hours, the LPS-stimulated mice were intraperitoneally challenged with 1 × 10⁹ CFU of K. pneumoniae KPC160111 or KPC160132. Peritoneal cells were harvested by injecting 5 mL of PBS at 1.5 h post-bacterial challenge, and the supernatants were collected for cytokine/chemokine analysis. After RBC lysis and washing, peritoneal cells were treated with Mouse Seroblock FcR (Bio-Rad Laboratories # BUF041) for 20 min at 4°C to block the Fc receptors and then incubated with rat anti-mouse fluorescent antibodies to comparatively analyze the distribution of the myeloid cells in response to K. pneumoniae challenge. The antibodies used were PE-conjugated anti-F4/80 (eBioscience #12-4801-82), FITC-conjugated anti-CD11b (eBioscience #11-0112-82), FITC-conjugated anti-CD86 (eBioscience #11-0862-82), and PE-eFluor 610-conjugated anti-CD163 (eBioscience #61-1631-82). After incubation at 4°C for 1 h, cells were washed and analyzed with a BD FACS Canto II cytometer (Becton Dickinson). Flow cytometry data were analyzed with FlowJO v10.7 (Becton Dickinson).

A five-color flow cytometric analysis was performed to determine the distribution of peripheral blood leukocytes. Antibodies against mouse leukocyte surface antigens were added to 100 μL aliquots of whole blood. The antibodies used to detect different subsets of 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, FITC-conjugated anti-Ly6G (BD Biosciences, San Jose, CA, USA) for neutrophils, APC-conjugated anti-CD3ε (eBioscience) for T cells, and Pacific blue-conjugated anti-CD19 (Biolegend) for B cells. Antibodies were used at the concentration recommended by manufacturer. After a 30 min incubation at 4°C in the dark, red blood cells were lysed, and cells were suspended in staining buffer and then analyzed with a FACS Canto II flow cytometer (BD Biosciences). CD45-positive cells were gated, and results are presented as a percentage of specific CD-marker-expressing cells in blood leukocytes. Representative flow cytometry plots are shown in Figure 2(a).

eWAT of WT and Redd1^−/− mice fed HFD for 16 weeks were minced, chopped, and digested with collagenase II (1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA; #C2674) in Krebs-Ringer HEPES Buffer for 15 min at 37 °C with gentle shaking. After passing cells through a 200 μm cell strainer and centrifugation at 1000× g for 10 min, the floating cells were collected as the mature adipocytes, and the pelleted cells were obtained as the SVF cells. The adipocytes were washed twice with DMEM to remove cell debris by centrifugation as above. The SVF cells were resuspended in erythrocyte lysis buffer and incubated at room temperature for 5 min. The erythrocyte-depleted SVF cells were centrifuged at 500 × g for 5 min and the pellet was resuspended and used for positive selection of adipose tissue macrophages. Briefly, the cells were labeled with the CD11b Microbead (10 μl/10⁷ cells, #130-049-601, Miltenyi Biotec Inc., Bergisch Gladbach, Germany) and passed through magnetic-activated cell sorting (MACS) separation column (Miltenyi Biotec). After washing twice, CD11b⁺ cells were removed from the column by washing twice with 2 ml MACS buffer away from the magnetic field. The purity of the isolated cells was determined using FITC-conjugated anti-CD11b (#11-0118-42, eBioscience, San Diego, CA) and PE-conjugated anti-F4/80 antibodies (#15-4801-82, eBioscience) and was greater than 95%.