Ly6c percp cy5

The magnetically separated muscle-derived CD45⁺ cells were stained with a combination of Alexa Fluor 488-conjugated anti-F4/80 antibody (MF48020, Invitrogen, Carlsbad, USA) and Alexa Fluor 647 conjugated anti-Ly6G/Ly6C (GR-1) antibodies (108418, BioLegend, San Diego, USA) at room temperature for 15 minutes. Cells were gated based on their forward- and side-scatter characteristics. Macrophages were gated as GR-1 negative and F4/80 positive, while neutrophils as F4/80-negative and GR-1-positive cells. F4/80-positive macrophages were also analyzed for Ly6C, CD206 or MHCII expressions following staining with Ly6C PerCP-Cy5.5 (128012, BioLegend, San Diego, USA), CD206-PE (141705, BioLegend, San Diego, USA) or MHCII-FITC (107605, BioLegend, San Diego, USA) antibodies, respectively. Fluorescent intensity was detected with a Becton Dickinson FACSCalibur instrument.

Peripheral blood mononuclear cells, peritoneal cells or lamina propria cells were taken and blocked in 5% fetal bovine serum for 20 min and then stained with CD45-FITC, CD11b-PE, CCR2-APC, Ly6C-PerCP Cy5.5, Ly6G-PerCP Cy5.5, F4/80-APC, or/and CD11c-APC (BioLegend) at 4 °C for 30 min. Data were acquired from FACS caliber (BD Biosciences) and analyzed using FlowJo 7.6 (TreeStar).

Circulating monocytes were labeled with 5-ethynyl-2′-deoxyuridine (EdU) to examine monocyte recruitment into atherosclerotic lesions, as described previously (35 (link)). Mice were injected intraperitoneally with 250 μl containing 1 μM EdU in saline (Invitrogen, Waltham, MA) two days before the sacrifice. To measure monocyte labeling efficiency, we examined EdU+ monocytes in circulation 48 h after injection by flow cytometry. We incubated blood samples with red blood cell lysis buffer (BioLegend, San Diego, CA) and stained them with primary antibodies antimouse CD45-FITC (Clone 30-F11; BioLegend), CD115-APC (Clone AFS98; BioLegend), and Ly6c-PerCP/Cy5.5 (Clone RB6-8C5; BioLegend) to identify circulating monocytes. Cells were permeabilized, and EdU was detected using Click-IT EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen). Flow cytometry was performed in LSR II analyzer (B.D. Biosciences, Franklin Lakes, NJ), and data were analyzed using FCS Express 5.01.0082 (De Novo Software, Pasadena, CA; https://denovosoftware.com/).

Mice were perfused transcardially under isoflurane anesthesia with phosphate-buffered saline (PBS) to flush vessels before euthanasia. After euthanasia, eyes were enucleated into ice-cold phosphate-buffered saline (PBS). The corneas, lenses and hyaloid vasculatures were removed and the retinas isolated immediately. To dissociate retinas into single cells, each retina was incubated in 1 ml of papain dissociation solution (Roche Diagnostics GmbH, REF#10108014001, Mannheim, Germany) prepared according to manufacturer’s protocol for 30 min in a 37 °C water bath. Single cell suspensions were washed with FACs buffer on ice and then incubated with primary antibody cocktails for 45 min at 4 °C in the dark. Antibodies used included CD45 Apc-eFluor780 (Invitrogen, Cat#47-0451-82, Waltham, MA, USA), Ly6C Percp/Cy5.5 (Biolegend, Cat#128012, San Diego, CA, USA), Ly6G (BV605 Biolegend, Cat#127639), CD11b PE-CF594 (BD Biosciences, Cat#562287, Franklin Lakes, NJ, USA), CD31 PE (Invitrogen, Cat#2114546) and Fixable viability Dye eFluor506 (Invitrogen, Cat# 65-0866-14). The cells were then washed with FACs buffer, resuspended and analyzed using BD FACSCelesta flow cytometer (BD Biosciences) and FlowJo™ v10.8 Software (BD Life Sciences, Franklin Lakes, NJ, USA). Gating of the different cell populations was performed as previously published [23 (link)].

Nine-week-old male BALB/c mice were used for these experiments. Animals were anaesthetized by isoflurane (Forene). After abdominal incision, the cecum was ligated, punctured with a gauge needle (25G), and a small amount of fecal matter was released. For the sham group, after abdominal incision, the cecum was manipulated but was neither ligated nor punctured. After the cecum was returned to the abdomen, the abdominal cavity was closed in two layers and the mice were resuscitated with 30 ml kg⁻¹ body weight of saline (0.9% NaCl) administered subcutaneously. LCC-12 (0.3 mg kg⁻¹, intraperitoneal injection) was administered at 4 h, 24 h, 48 h, 72 h and 96 h following CLP creation. Mortality incidence was monitored every 2 h up to 120 h (except from 10 pm to 6 am) after CLP creation. Dexamethasone was administered intraperitoneally at 1 mg kg⁻¹ 5 min before CLP creation. ICP-MS experiments were conducted on SPMs as described in ‘ICP-MS’. Tissue-specific data were normalized against dry weight. The sorting of SPMs was done using the following antibodies: CD11b-Pacific Blue (BioLegend, 101224), F4/80-PE (TONBO, TNB50-4801-U100), Ly6C–PerCP/Cy5.5 (BioLegend, 128012) and Ly6G–AF647 (BioLegend, 127610). The sorted SPMs corresponded to CD11b⁺F4/80^intLy6C⁻Ly6G⁻ cells.