Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis

Abstract

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response.

Maternal circulating leukocytes display early chemotactic responsiveness during late gestation

“…The Boyden chamber migration assay is a very sensitive technique for assessing leukocyte responsiveness to chemotactic signals [20]. To our knowledge, this is the first time it has been used in an animal study with the purpose of delineating temporal events in the birth cascade.…”

“…The chemotaxis assay was performed using a modified validated Boyden chamber assay (AP48, Neuro Probe, USA) [20]. First, we investigated the onset of peripheral leukocyte migration or chemotaxis during gestation in response to uterine and cervical extracts obtained at term (GD22).…”

“…Chambers were incubated for 120 min at 37°C in humidified air containing 5% CO 2 . Incubation time was previously established by performing incubation time curves [20]. Afterwards, chemoattracted leukocytes were removed from the lower compartment and centrifuged at 500 × g for 5 min at room temperature.…”

“…The phenotype of leukocytes was analyzed within the CD45 + and CD3 + region, respectively. Granulocytes were identified as CD45 + OX43 - CD45R - CD161 - CD3 - by using the parameters of FS, SS and CD45 [20]. …”

Normal and Premature Rupture of Fetal Membranes at Term Delivery Differ in Regional Chemotactic Activity and Related Chemokine/Cytokine Production

“…Multiple explants of 1 cm 2 tissue were obtained from each fetal membrane zone: the RZ was labeled with Gentian violet; the middle zone (MZ), halfway between the RZ and the placental edge at least 10 to 12 cm from the RZ; and from the PZ, comprising the 1 to 2 cm of thickened fetal membranes next to the edge of the placenta. 26 The entire fetal membranes were used and the total number of explants varied between patients (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and depended on the length of the RZ and the distance between the zone and placental edge. Explants were washed carefully and immediately placed in sterile saline solution to eliminate visible blood clots, maintaining the integrity of the membranes.…”