Evaluation of directed and random motility in microslides Assessment of leukocyte adhesion in flow chambers
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1 Evaluation of directed and random motility in microslides Motility experiments in IBIDI microslides, image acquisition and processing were performed as described. PMN, which ended up in an angle < 180 towards the segment of upward gradient were indicated as toward up-gradient segment, PMN, which moved into any-direction were indicated as towards any direction. Assessment of leukocyte adhesion in flow chambers Heparinized blood was drawn from healthy volunteers and erythrocytes were lysed by addition of 10 volume of 0.83% NH 4Cl, 0.1% KHCO3 and 1 mm EDTA in deionized water (ph 7.4). Cells were pelleted (300 g, 7 min) and resuspended in HBSS/0.25% BSA to a concentration of 500,000/ml. IBIDI µ-slides VI were coated with BSA (5%, Sigma) or MPO (20 µg/ml, Planta Natural Products) for 12 hours at 4 C. The slides were washed 6 with PBS and filled with 5% BSA in PBS to block unspecific binding sites. The slides were perfused with the leukocyte suspension starting with a shear stress of 0.5 dyn/cm², which was decreased to 0.25 dyn/cm² after 1 min. Cell flow was observed with an inverted microscope (Zeiss Axiovert 200, 20 objective) and recorded with a DVD recorder (Panasonic, LQ-MD800E) with CapImage Software (CapImage 8.5, Dr. Heinrich Zeintl, Heidelberg). Adherent cells were counted after 4 min of perfusion with 0.25 dyn/cm². Quantification of MPO after hepatic ischemia/reperfusion Ischemia and reperfusion in WT mice was performed as described. One group of animals was anaesthetized after 2 hours of reperfusion and blood (0.5 ml) was drawn from the portal vein into EDTA rinsed syringes. Mice were sacrifized thereafter. Otherwise mice were anaesthetized after 20 hours of reperfusion and the livers were flushed with 3 ml of PBS via the portal vein. The liver was excised and snap frozen in liquid nitrogen or embedded in OCT compound prior to freezing at 80 C. MPO in liver tissue was quantified with an ELISA (Hycult Biotechnology) according to the manufacturer s instruction. OCT samples were cut to 3 µm sections, fixed with 3.7 % formaldehyde, permeabilized with Triton X-100, blocked with 10% goat serum and incubated with primary antibodies to mouse Ly6G (rat IgG, 1:40, Hycult Biodiagnostics) and to mouse MPO (rabbit IgG, Thermo, 1:100, 1 h, RT). Sections were treated with secondary antibodies Alexa 488 to rabbit IgG and Alexa 594 to rat IgG (1:100, 1 h, RT, Molecular Probes) and nuclei were stained with Dapi. Images were acquired with a Leica fluorescence microscope (DMLB) with an oil immersion objective ( 20). Immunofluorescence staining of MPO in cremaster muscle whole mounts Murine TNF-α or saline was injected to the scrotum of WT mice as described. After two hours a catheter was inserted into the carotid artery and an antibody to murine MPO (60 µg, rabbit IgG, Thermo) was injected. After 10 min the V. cava inferior was incised and 5 ml of PBS were flushed via the carotid catheter. The cremaster muscle was excised and processed as described. Quantification of Mac-1 integrin expression by flow cytometry Blood was drawn from WT and Mpo mice to EDTA rinsed syringes. Where indicated samples were treated with murine TNF-α (50 ng/ml) for 30 min at RT. Erythrocytes were lysed by addition of 10 volume of 0.83% NH4Cl, 0.1% KHCO3 and 1 mm EDTA in deionized water (ph 7.4), washed in PBS and resuspended in 3.7% formaldehyde. To block unspecific binding sites the cells were incubated with 10% mouse serum in PBS for 15 min at 4 C. The following
2 antibodies were used: PerCpCy5.5-conjugated anti-cd11b, PE-conjugated anti-ly6g, V450- conjugated anti-ly6g/c, V500-conjugated anti-cd3 (BD). After repeated washing the cells were analyzed with a BD Biosciences LSR II system using FACS Diva software.
3 Table S1. Microcirculatory parameters of experimental groups following intrascrotal injection of TNF-α. Vessel diameter, wall shear rate, and systemic leukocyte counts are presented as mean ± s.e.m. of all investigated vessels. Mouse strain Treatment Animals (n) Vessels (n) Diam (µm) Wall Shear Rate (s-1) Syst. WBC (cells/µl) C57BL/6 TNF-α ± ± ± 303 MPO-/- TNF-α ± ± ± 167 Table S2. Microcirculatory parameters of experimental groups following systemic injection of indicated substances. Vessel diameter, wall shear rate, and systemic leukocyte counts are presented as mean ± s.e.m. of all investigated vessels. Mouse strain Treatment Animals (n) Vessels (n) Diam (µm) Wall Shear Rate (s-1) Syst. WBC (cells/µl) C57BL/6 Saline ± ± ± 306 C57BL/6 MPO ± ± ± 235 C57BL/6 MPO Q91T ± ± ± 214 MPO-/- MPO ± ± ± 228
4 A B Figure S1. PMN motility in vitro. PMN motility was evaluated in Ibidi microslides. (A) Use of plasma instead of HBSS buffer did not affect MPO-directed PMN motility (n=3). (B) Eosinophil peroxidase (EPO) induced PMN-motility to a similar extent as MPO (n=3). Bars represent means, error bars indicate s.e.m., ***p<0.001 Figure S2. Random and directed motility in microslides. Percentage of cells moving into any direction (chemokinesis or random migration) vs. moving towards the
5 increasing gradient (chemotaxis) is shown (n=9-34, one-way ANOVA p<0.001) Bars represent means, error bars indicate s.e.m. *p<0.05, **p<0.01, ***p< Figure S3. Adherent PMN in flow chambers. Number of adherent PMN per field of view (fov, 20x objective) in BSA or MPO coated flow chambers after 4 minutes of flow with shear stress of 0.25 dyn/cm 2 is depicted (n=3 independent experiments with 3-6 flow chamber slides of each condition, respectively, Student s unpaired T-test). Bars represent means, error bars indicate s.e.m. *p<0.05.
6 A B C Figure S4. MPO accumulation upon hepatic ischemia and reperfusion in WT mice. (A) Plasma MPO levels of blood drawn from the portal vein after 90 minutes of ischemia and 2 hours of reperfusion or sham surgery (n=3-4, Student s unpaired T-test) *p<0.05. (B) 90 minutes of ischemia with subsequent 20 hours of reperfusion provoked MPO accumulation in hepatic tissue (n=6-10). Bars represent means, error bars indicate s.e.m. (C) Hepatic sections of WT mice after 90 minutes of ischemia with subsequent 20 hours of reperfusion are shown (αmpo green, AlexaFluor 488; αly6g (PMN) red, AlexaFluor 594,
7 dapi blue, x 200, captured with Retiga 1300 CCD camera mounted on Leica DMLB fluorescence microscope by ivision v4.0). Figure S5. Immunofluorescence staining of MPO in cremasteric postcapillary venules. MPO (αmpo, green, AlexaFluor 488) was detected intraluminally 2 h after intrascrotal (i.sc.) injection of saline or TNF-α in WT mice (blue = dapi; scale bar 30µm; captured with Retiga 1300 CCD camera mounted on Leica DMLB fluorescence microscope by ivision v4.0).
8 A B C Figure S6. Systemic leukocyte counts and PMN integrin expression of WT and Mpo -/- mice. (A) Number of leukocytes from venous blood of untreated WT and Mpo -/- mice (n=5) was assessed. (B) Mac-1 integrin expression on the surface of WT and Mpo -/- mice PMN was evaluated by flow cytometry in unstimulated and TNF-α treated whole blood. Representative histograms (Mac-1 fluorescence intensity, log scale) and (C) means of relative fluorescence units (RFU) are depicted (n=3 animals, one-way ANOVA p<0.001). Error bars indicate s.e.m. **p<0.01, ***p<0.001.
9 A B C Figure S7. PMN recruitment in cremaster muscle venules and arterioles (A) The number of adherent leukocytes in cremaster muscle postcapillary venules was assessed before (basal, white bars) and after (post injection, black bars) systemic injection of active MPO (20 µg) in WT (15 vessels in 3 mice) and Mpo -/- mice (13 vessels in 3 mice, oneway ANOVA p<0.001). *p<0.05, ***p<0.001 (B) Number of adherent leukocytes in postcapillary venules (n=19) and arterioles (n=15-18) of cremaster muscle whole-mounts after systemic injection of active MPO, catalytically inactive Q91TMPO and saline was counted (3 animals per group, one-way ANOVA p<0.001). Bars represent means, error bars indicate s.e.m. ***p< (C) Representative images of arterioles and venules in the
10 Giemsa stained cremaster muscle after systemic injection of MPO (scale bar 40 µm, Zeiss upright microscope with an oil immersion objective (40 x, 1.3 numerical aperture)). Figure S8. Erythrocyte motility in vitro. Erythrocyte (RBC) motility was evaluated in Ibidi microslides. RBCs displayed directed motility towards MPO (n=3). Bars represent mean, error bars indicate s.e.m.
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