Figure 2.
Figure 2. Rap1b−/− neutrophils exhibit enhanced chemokinesis and chemotaxis. (A) Analysis of neutrophil migration using time lapse video microscopy in a gradient of fMLP in Zigmond chamber. Cell trajectory analysis; the schema represents the migration trajectory of cells moving up fMLP gradient for 20 min. Speeds (Sp = µm/min) of migration are indicated at the bottom. Mean ± SD; n = 60 cells. Scatter plot of straightness of migration from 30 individual cells, representative of 3 independent experiments. Bar graph represents the percentage of cells exhibiting frequent changes in direction. Data are from 80 cells (mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; 3 independent experiments). (B and C) Chemokinesis (uniform chemokine concentration) and chemotaxis (chemokine gradient) analysis of neutrophils using a Boyden chamber with 1 µM fMLP, 100 nM MIP2, or differential concentration of chemokines. The histogram represents the number of migrated neutrophils per field using a 40× objective (mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; representative of 3 independent experiments). (D) Neutrophil migration using Transwell-coated with fibrinogen (Fg) or endothelial cells (HUVECs and bEND.3) grown on Transwell filters in uniform concentration or in a gradient of 10 µM fMLP. Histogram represents the total number of migrated neutrophils recovered from the bottom well (mean ± SD; n = 3; **, P < 0.01; ***, P < 0.001; using unpaired Student’s t test; 3 independent experiments). (E) Adhesion analysis of WT and Rap1b−/− neutrophils that were stimulated with fMLP and plated on diverse integrin ligands. Expression of integrins on WT and Rap1b−/− neutrophils with or without fMLP stimulation, as quantified using flow cytometry (mean ± SD; n = 3 independent experiments). (F) Images and quantification of spreading from minimum 50 WT and Rap1b−/− neutrophils that were plated on CD11b-coated plates without fMLP. Bar, 10 µm. Mean ± SD; n = 3 independent experiments **, P < 0.01. (G) Superoxide generation analysis in suspension by using flow cytometry and DCF-DA probe. Mean ± SD; n = 3 independent experiments; **, P < 0.01 (unpaired Student’s t test). (H) Superoxide generation kinetics from neutrophils in 96-well plates under adherent condition after stimulation with different concentration of fMLP or PMA. Chemiluminescence of L012 was measured for 20 min using GloMax-96 Microplate Luminometer (Promega). n = 3 independent experiments.

Rap1b−/− neutrophils exhibit enhanced chemokinesis and chemotaxis. (A) Analysis of neutrophil migration using time lapse video microscopy in a gradient of fMLP in Zigmond chamber. Cell trajectory analysis; the schema represents the migration trajectory of cells moving up fMLP gradient for 20 min. Speeds (Sp = µm/min) of migration are indicated at the bottom. Mean ± SD; n = 60 cells. Scatter plot of straightness of migration from 30 individual cells, representative of 3 independent experiments. Bar graph represents the percentage of cells exhibiting frequent changes in direction. Data are from 80 cells (mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; 3 independent experiments). (B and C) Chemokinesis (uniform chemokine concentration) and chemotaxis (chemokine gradient) analysis of neutrophils using a Boyden chamber with 1 µM fMLP, 100 nM MIP2, or differential concentration of chemokines. The histogram represents the number of migrated neutrophils per field using a 40× objective (mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; representative of 3 independent experiments). (D) Neutrophil migration using Transwell-coated with fibrinogen (Fg) or endothelial cells (HUVECs and bEND.3) grown on Transwell filters in uniform concentration or in a gradient of 10 µM fMLP. Histogram represents the total number of migrated neutrophils recovered from the bottom well (mean ± SD; n = 3; **, P < 0.01; ***, P < 0.001; using unpaired Student’s t test; 3 independent experiments). (E) Adhesion analysis of WT and Rap1b−/− neutrophils that were stimulated with fMLP and plated on diverse integrin ligands. Expression of integrins on WT and Rap1b−/− neutrophils with or without fMLP stimulation, as quantified using flow cytometry (mean ± SD; n = 3 independent experiments). (F) Images and quantification of spreading from minimum 50 WT and Rap1b−/− neutrophils that were plated on CD11b-coated plates without fMLP. Bar, 10 µm. Mean ± SD; n = 3 independent experiments **, P < 0.01. (G) Superoxide generation analysis in suspension by using flow cytometry and DCF-DA probe. Mean ± SD; n = 3 independent experiments; **, P < 0.01 (unpaired Student’s t test). (H) Superoxide generation kinetics from neutrophils in 96-well plates under adherent condition after stimulation with different concentration of fMLP or PMA. Chemiluminescence of L012 was measured for 20 min using GloMax-96 Microplate Luminometer (Promega). n = 3 independent experiments.

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