Figure 7.
Figure 7. Extravasation, chemotaxis, and phagocytosis capacity of neutrophils in TG2+/+ and TG2–/– mice. (A) Accumulation of neutrophils in the peritoneum when wild-type and TG2-deficient mice were injected intraperitoneally with 1 mL 10% yeast extract. At 4 hours, mice were injected intraperitoneally with 3 mL RPMI-1640 medium and total lavage fluid was withdrawn. To create a monolayer of PMN cells, the granulocytes were allowed to adhere for 30 minutes, followed by gentle washing of the monolayer with culture medium to remove nonadherent cells. Cell number was evaluated on 25 to 30 fields of view seen through the eyepieces of the microscope. (B) Changes of neutrophil counts in circulation 4 hours after injection of 1 mL 10% yeast extract. Mice were killed and their blood samples were collected by cardiac puncture and analyzed for neutrophil counts by fluorescence-activated cell sorting (FACS). In panels A and B, the data represent mean ± SD from 6 to 8 mice per group. (C) In vitro evaluation of chemotaxis of TG2+/+ and TG2–/– neutrophils. Wild-type and TG2–/– peritoneal neutrophils were isolated and kept in serum-free medium overnight and then added into Matrigel Invasion upper chambers and allowed to migrate into the lower chambers containing RPMI 1640 supplemented with 10% mouse serum and 100 nM fMLP. The number of neutrophils migrating through the chambers was determined by photographing both the upper and bottom sides of membranes at the beginning and end of migration. The migration time was 4 hours. (D) In vitro evaluation of TG2+/+ neutrophil chemotaxis in the presence or absence of MDC. Peritoneal lavage neutrophils were kept in serum-free medium in the presence or absence of MDC overnight and then added into Matrigel Invasion upper chambers and allowed to migrate into the lower chambers. Where it is indicated, the lower chambers contained RPMI 1640 medium with or without the addition of 10% mouse serum, 100 nM fMLP, and 15 μM MDC. The number of neutrophils that migrated through the chambers was determined as described previously in panel C, with the difference that the migration time was 8 hours. (E) Percentage of phagocytosing neutrophil granulocytes. Mixtures of heparinized whole blood and FITC-labeled E coli were incubated at 37°C and 0°C, respectively, the fluorescence of the attached bacteria on the cell surface was quenched, and then erythrocytes were lysed. Fluorescing cells out of a total of 10 000 granulocytes were counted by a FACSCalibur instrument and expressed in percentage of total cell number. Each experimental group included 2 to 4 mice, and each individual experiment was performed 2 or 3 times in duplicates. Bars depict the means ± SD.

Extravasation, chemotaxis, and phagocytosis capacity of neutrophils in TG2+/+ and TG2–/– mice. (A) Accumulation of neutrophils in the peritoneum when wild-type and TG2-deficient mice were injected intraperitoneally with 1 mL 10% yeast extract. At 4 hours, mice were injected intraperitoneally with 3 mL RPMI-1640 medium and total lavage fluid was withdrawn. To create a monolayer of PMN cells, the granulocytes were allowed to adhere for 30 minutes, followed by gentle washing of the monolayer with culture medium to remove nonadherent cells. Cell number was evaluated on 25 to 30 fields of view seen through the eyepieces of the microscope. (B) Changes of neutrophil counts in circulation 4 hours after injection of 1 mL 10% yeast extract. Mice were killed and their blood samples were collected by cardiac puncture and analyzed for neutrophil counts by fluorescence-activated cell sorting (FACS). In panels A and B, the data represent mean ± SD from 6 to 8 mice per group. (C) In vitro evaluation of chemotaxis of TG2+/+ and TG2–/– neutrophils. Wild-type and TG2–/– peritoneal neutrophils were isolated and kept in serum-free medium overnight and then added into Matrigel Invasion upper chambers and allowed to migrate into the lower chambers containing RPMI 1640 supplemented with 10% mouse serum and 100 nM fMLP. The number of neutrophils migrating through the chambers was determined by photographing both the upper and bottom sides of membranes at the beginning and end of migration. The migration time was 4 hours. (D) In vitro evaluation of TG2+/+ neutrophil chemotaxis in the presence or absence of MDC. Peritoneal lavage neutrophils were kept in serum-free medium in the presence or absence of MDC overnight and then added into Matrigel Invasion upper chambers and allowed to migrate into the lower chambers. Where it is indicated, the lower chambers contained RPMI 1640 medium with or without the addition of 10% mouse serum, 100 nM fMLP, and 15 μM MDC. The number of neutrophils that migrated through the chambers was determined as described previously in panel C, with the difference that the migration time was 8 hours. (E) Percentage of phagocytosing neutrophil granulocytes. Mixtures of heparinized whole blood and FITC-labeled E coli were incubated at 37°C and 0°C, respectively, the fluorescence of the attached bacteria on the cell surface was quenched, and then erythrocytes were lysed. Fluorescing cells out of a total of 10 000 granulocytes were counted by a FACSCalibur instrument and expressed in percentage of total cell number. Each experimental group included 2 to 4 mice, and each individual experiment was performed 2 or 3 times in duplicates. Bars depict the means ± SD.

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