Anti f4 80 efluor450 mab clone bm8

After 6 h of i.p. stimulation with CCL2, mice were sacrificed and their peritoneal cavity was washed with 10 ml of ice-cold PBS as described previously. The total number of leukocytes recovered from the peritoneal lavage fluid was analyzed by using a Coulter A C T counter (Coulter Corp.). Samples were then labeled with anti-CD45 APC-Cy7 mAb (clone 30-F11; BD Bioscience), anti-CD11b FITC mAb (clone M1/70; eBioscience, San Diego, CA, US), anti-GR-1 PE mAb (clone RB6-8C5; eBioscience), anti-CD115 APC mAb (clone AFS98; eBioscience), and anti-F4/80 eFluor450 mAb (clone BM8; eBioscience) for 30 minutes on ice. Erythrocytes were lysed with lysing solution (1:10; BD FACS lysing solution; BD Bioscience). After two washing steps, leukocytes were resuspended in 250 μl PBS.
Using flow cytometry (Gallios; Beckman Coulter Inc, Brea, CA, USA), myeloid leukocytes were detected by expression of CD45 and CD11b as well as by the absence of F4/80. Therefore, neutrophils were identified by high expression of Gr-1 and low expression of CD115, iMOs by high expression of Gr-1 and CD115, and resident monocytes by high expression of CD115 as well as by low expression of Gr-1.

To study the effect of VN-deficiency on immune cell infiltration of liver, kidneys, and lungs of mice with severe systemic inflammation, organs were harvested 6 h after intraperitoneal injection of LPS (see above) and homogenized. The platelet count as well as the total white blood count of each organ homogenate was determined using a ProCyte Dx cell counter (IDEXX Laboratories, Westbrook, ME, USA). After incubation with anti-CD45-APC/Cy7 mAb (clone 30-F11; BD Bioscience), anti-CD11b-FITC mAb (clone M1/70; eBioscience, Waltham, Massachusetts, USA), anti-GR-1-PE mAb (clone RB6-8c5; eBioscience), anti-F4/80-eFluor 450 mAb (clone BM8; eBioscience) for 30 min on ice, lysis of erythrocytes was performed using lysing solution (BD Biosciences) for 10 min at room temperature. After washing, immunostained cells were analyzed on a flow cytometer (Gallios flow cytometer; Beckman Coulter Inc, Brea, California, USA). Approximately 20,000 gated events were collected in each analysis and isotype-matched controls were used in all experiments. Data analysis was performed using FlowJo software (Becton, Dickinson and Company). Absolute immune cell numbers were calculated using the white blood count and the relative share of the respective immune cell subset as assessed by flow cytometry.