The Effect of CO₂ and HEPES on Bovine Neutrophil Migratory Function

Nebenhaus, Regan
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University of Delaware
The objective of this study was to investigate the effects of incubation with CO₂ and HEPES on bovine neutrophil chemotaxis assays. On each sampling day, blood was collected from four lactating cows (4-163 days in milk; n=12 cows total). Neutrophils were isolated and adjusted to 2 × 10^6 cells/mL in Hank’s Buffered Salt Solution containing 5% fetal bovine serum. For the chemotaxis assay a 48 well chamber was used (Neuro Probe Inc., Gaithersburg, MD). The bottom wells of the chambers contained 28 μl of media (with or without HEPES) supplemented with either 100 ng/mL of interleukin-8 (IL-8) or 50 ng/mL of complement component 5a (C5a). A five μm polycarbonate membrane (Neuro Probe Inc., Gaithersburg, MD) was used. The top wells of the chamber were then filled with 50 μl of PMN suspension. The chambers were incubated for 60 minutes at 37℃ either with or without 5% CO₂. Neutrophil migration was determined by adherence to the membrane and concentration in the bottom wells of the chemotaxis chamber. Chemotaxis toward IL-8 and C5a was higher than toward controls for all measures. Chemotaxis toward IL-8 was not different from chemotaxis toward C5a. When chemotaxis was assessed by adherence to the membrane, the presence of CO₂ during incubation decreased neutrophil (PMN ) migration, but concentration of PMN in the bottom wells of the chamber increased in the presence of CO₂. Inclusion of HEPES in the assay media increased concentration of PMN in the bottom wells but did not affect adherence. There was a t. An interaction was found between HEPES and chemoattractant on Con of migrated PMN (P=0.002). When HEPES was present there was an increase in PMN migration to negative control, C5a, and positive control wells compared to when HEPES was not present. However, this HEPES-induced increase was significantly higher for migration of PMN to IL-8. There was an interaction between HEPES and CO₂ on Ad of migrated PMN (P=0.0001). When CO2 was present HEPES increased Ad of PMN. However when CO₂ was not present there was no effect of HEPES on Ad of PMN. In conclusion when PMN were incubated with CO₂ there was a decrease in function observed by Adhesion measurements and concentration measurements, however more work is need to further understand this relationship.