Centred to co-ordinate (0,0) to better view the distance travelled. A circular

Centred to co-ordinate (0,0) to better view the distance travelled. A circular horizon distance was set and the number of cells from the total population that reached the horizon distance was monitored. The random speed and the persistence in the random motion was calculated and compared. Mathematical analysis was carried out using Mathematica 6.0TM 69-25-0 chemical information workbooks [17]. P-values less 0.05 were accepted as statistically significant. To study chemotaxis, cells were seeded on acid washed coverslips at a density of 26104 cells/ml in macrophage growth medium and incubated overnight. Following incubation cells were starved of CSF-1 in macrophage starve medium for 8 hours. The coverslips were then mounted onto Dunn chemotaxis chambers as previously described [18] using recombinant murine CSF-1 (30 ng/ml) as the chemoattractant. Cell images were collected and analysed as described above.ImmunofluoresenceBMMs were seeded on glass coverslips at 26105 cells per coverslip and maintained in macrophage growth or macrophage starve medium followed by CSF-1 stimulation as indicated. Cells were washed with PBS, fixed with 4 paraformaldehyde permeabilised and stained for actin using phalloidin-FITC. The actin images were collected on IX71 Olympus microscope and cell images were analysed using ImageJ.ImmunoblottingCells were seeded onto 6 well plates and maintained or CSF-1 deprived as outlined above. Following stimulation cells were lysed as previously described [17] and lysates subjected to acrylamide gel electrophoresis as previously described [17]. Protein membranes were blocked and probed with primary and secondary antibodies as indicated. The blots were developed using PierceH ECL Western Blotting Substrate Kit (Thermo Scientific). The autoradiograph were scanned and band densities were quantified with Kinetic Imaging software to obtain the ratio of AZ-876 phosphorylated protein to total protein.Isolation and Culture of Mouse Primary Bone Marrow Derived MacrophagesThe murine femoral bones were harvested after the mice were culled using terminal anaesthesia. All the surrounding tissue on the bone was removed and the bone pierced at both ends with a 21-gauge needle. The bone marrow was flushed out of the bone with macrophage starve medium (RPMI 1640 with L-glutamine, 1 Essential amino acids, 1 sodium pyruvate, 1 P+S, 10 FCS and 0.5 bmercaptoethanol). Cells were then centrifuged and the pellet resuspended in macrophage starve medium. The cells were then counted and 26105 cells/ cm2 seeded onto 10 cm petri dishes for 3 days in macrophage growth media (starve medium plus M-CSF1 at 30 ng/ml). After 3 days the non-adherent population of cells containing the monocytes was removed. The cells were centrifuged, resuspended and counted. The cells were then seeded onto 6-cm bacterial culture plates at a density of 105 cells/mL. The cells are incubated for a further 5 days in the presence of M-CSF-1 as above. The differentiated BMMs become adherent and were harvested on day 5 for experimentation.Results Nox2KO Macrophages have an Increased Spread AreaWe were interested in establishing whether Nox2 plays a role in the infiltration of macrophages at sites of inflammatory response, such as those that are thought to be associated with tissue repair or conditions such as atherosclerosis. Many of the signalling pathways that regulate cellular migration are the same as those controlling cellular morphology. Therefore we first analysed the cell morphology of WT and macrophages.Centred to co-ordinate (0,0) to better view the distance travelled. A circular horizon distance was set and the number of cells from the total population that reached the horizon distance was monitored. The random speed and the persistence in the random motion was calculated and compared. Mathematical analysis was carried out using Mathematica 6.0TM workbooks [17]. P-values less 0.05 were accepted as statistically significant. To study chemotaxis, cells were seeded on acid washed coverslips at a density of 26104 cells/ml in macrophage growth medium and incubated overnight. Following incubation cells were starved of CSF-1 in macrophage starve medium for 8 hours. The coverslips were then mounted onto Dunn chemotaxis chambers as previously described [18] using recombinant murine CSF-1 (30 ng/ml) as the chemoattractant. Cell images were collected and analysed as described above.ImmunofluoresenceBMMs were seeded on glass coverslips at 26105 cells per coverslip and maintained in macrophage growth or macrophage starve medium followed by CSF-1 stimulation as indicated. Cells were washed with PBS, fixed with 4 paraformaldehyde permeabilised and stained for actin using phalloidin-FITC. The actin images were collected on IX71 Olympus microscope and cell images were analysed using ImageJ.ImmunoblottingCells were seeded onto 6 well plates and maintained or CSF-1 deprived as outlined above. Following stimulation cells were lysed as previously described [17] and lysates subjected to acrylamide gel electrophoresis as previously described [17]. Protein membranes were blocked and probed with primary and secondary antibodies as indicated. The blots were developed using PierceH ECL Western Blotting Substrate Kit (Thermo Scientific). The autoradiograph were scanned and band densities were quantified with Kinetic Imaging software to obtain the ratio of phosphorylated protein to total protein.Isolation and Culture of Mouse Primary Bone Marrow Derived MacrophagesThe murine femoral bones were harvested after the mice were culled using terminal anaesthesia. All the surrounding tissue on the bone was removed and the bone pierced at both ends with a 21-gauge needle. The bone marrow was flushed out of the bone with macrophage starve medium (RPMI 1640 with L-glutamine, 1 Essential amino acids, 1 sodium pyruvate, 1 P+S, 10 FCS and 0.5 bmercaptoethanol). Cells were then centrifuged and the pellet resuspended in macrophage starve medium. The cells were then counted and 26105 cells/ cm2 seeded onto 10 cm petri dishes for 3 days in macrophage growth media (starve medium plus M-CSF1 at 30 ng/ml). After 3 days the non-adherent population of cells containing the monocytes was removed. The cells were centrifuged, resuspended and counted. The cells were then seeded onto 6-cm bacterial culture plates at a density of 105 cells/mL. The cells are incubated for a further 5 days in the presence of M-CSF-1 as above. The differentiated BMMs become adherent and were harvested on day 5 for experimentation.Results Nox2KO Macrophages have an Increased Spread AreaWe were interested in establishing whether Nox2 plays a role in the infiltration of macrophages at sites of inflammatory response, such as those that are thought to be associated with tissue repair or conditions such as atherosclerosis. Many of the signalling pathways that regulate cellular migration are the same as those controlling cellular morphology. Therefore we first analysed the cell morphology of WT and macrophages.