Gα_i-independent neutrophil recruitment in vivo requires PSGL-1 and CD44, all 3 SFKs, and Btk. (A) WT, PSGL-1^−/−, CD44^−/−, or PSGL-1^−/−/CD44^−/− mice received 4 μg of PTx intravenously 2 hours before injection with 1 mL of 4% thioglycollate intraperitoneally. After 4 hours, peritoneal cells were collected, and the number of neutrophils was measured by flow cytometry. (B) WT leukocytes were labeled with PKH67 or PKH26. SFK-deficient leukocytes were labeled with PKH26. Untreated or PTx-treated WT mice were injected intraperitoneally with thioglycollate. After 2 hours, they were injected intravenously with a 1:1 mixture of PKH26- and PKH67-labeled leukocytes. After another 2 hours, blood and peritoneal cells were collected, and the number of neutrophils labeled with each dye was measured by flow cytometry. Results are plotted as the ratio of PKH26-labeled neutrophils from the indicated genotype to PKH67-labeled WT neutrophils. (C) Untreated or PTx-pretreated WT or Btk^−/− mice were injected with thioglycollate intraperitoneally. After 4 hours, peritoneal cells were collected, and the number of neutrophils was measured by flow cytometry. (D) Competitive homing was measured as in panel B, except that Btk^−/− leukocytes were used instead of SFK-deficient leukocytes. The data represent the mean ± SEM from at least 3 experiments. *P < .01.