Fitc conjugated anti cd62l dreg 65

Surface antigen expression was examined with PMNs isolated at 9–680 kgs (10g, 20g, 47g, 756g each for 15 min) as well as with the 1g control group. Briefly, 200 µL of the isolated leukocyte-rich supernatant were laid into a 2 mL centrifugation tube; 590 µL of RPMI 1640 medium (PAN-Biotech GmbH, Aidenbach, Germany), 200 µL of autologous serum and 10 µL of 1 µM fMLP (Sigma Aldrich) were added. From this leukocyte-suspension, samples were withdrawn before incubation was started (t = 0) as well as after incubation for 22 h (t = 22) at 37 °C in a HeraCell™ 150i CO₂-Incubator (Thermo Fisher Scientific).
Expression of surface antigens was examined by immunostaining with PE-conjugated anti-CD11b (ICRF44, BioLegend, San Diego, CA, USA), FITC-conjugated anti-CD62L (DREG-65, BioLegend) and APC-conjugated anti-CD66b (G10F5, BioLegend). The immunostaining process involved two centrifugation steps with a combined g-time load of 209 kgs, which were both performed at 4 °C immediately before flow cytometric measurement.

After gelafundin sedimentation, the cell samples were incubated with or without lidocaine, bupivacaine and levobupivacaine. After 0.5 h, 3 h, 6 h and 12 h, an oxidative burst and an antigen analysis for CD11b, CD62L and CD66b were performed, which has been described in detail by Kupke et al. and Trabold et al. [26 (link),31 (link)]. Upon completion of the incubation period with the LAs, the burst was performed without stimulation and with stimulation by fMLP (10 µM, Sigma Aldrich, Steinheim, Germany) in combination with tumor necrosis factor alpha (TNF-α; 1 µg/mL, Thermo Fisher Scientific, Agawam, MA, USA), or with phorbol-12-myristate-13-acetate (PMA; 10 µM, Sigma Aldrich, Steinheim, Germany). ROS production was detected with DHR, which is converted to fluorescent rhodamine 123 by oxidation through reactive oxygen species. We used PE-conjugated anti-CD11b (ICRF44, BioLegend, San Diego, CA, USA), FITC-conjugated anti-CD62L (DREG-65, BioLegend, San Diego, CA, USA) and APC-conjugated anti-CD66b (G10F5, BioLegend, San Diego, CA, USA) to visualize the antigens. For detection, we used a FACS Calibur™ flow cytometer (BD corporate, Franklin Lakes, NJ, USA) and CellQuest Pro software™ (version 5.2, BD corporate, Franklin Lakes, NJ, USA).

The expression of surface antigens was examined by immunostaining with PE-conjugated anti-CD11b (ICRF44; BioLegend, San Diego, CA, USA), FITC-conjugated anti-CD62L (DREG65; BioLegend), and APC-conjugated anti-CD66b (G10F5; BioLegend). For this purpose, 20 µL of cell concentrate was mixed with 1 mL PBS and centrifuged at 425× g for 3 min. The supernatant was removed and 5 μL each of CD11b, CD62L, and CD66b anti-human antibodies were added and incubated at 4 °C for 15 min. The cell suspension was washed with 2 mL PBS; after centrifugation, the supernatant was removed again, and the cells were resuspended (200 µL PBS) and analyzed.