The effects of CO2 and HEPES buffer on in-vitro chemotaxis assays of bovine neutrophils
Neutrophil (PMN) chemotaxis can be measured in vitro with or without 5% CO2. The production of carbonic acid during incubation with 5% CO2 may reduce pH, possibly affecting cellular migratory function. The goals of this study were to determine whether incubation with 5% CO2 impacts PMN chemotactic function and if the addition of HEPES buffer affects response to CO2. Neutrophils were isolated from 12 lactating cows over 5 different test dates and cell concentration adjusted to 2×106 cells/mL. Media consisted of HBSS containing 5% FBS with or without the addition of 10 mM HEPES. The bottom wells of a 48-well chemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD) contained 28 µL of media supplemented with 50 ng/mL of complement component 5a (C5a) or 100 ng/mL of Interleukin-8 (IL-8). A 5 µm polycarbonate membrane separated the top and bottom wells. Neutrophil suspension (50 µL) was added to the top wells, and the chambers were incubated at 37°C for one hour, in the presence or absence of 5% CO2. Negative controls did not contain chemoattractant in either well while positive controls contained 50 ng/mL C5a in both top and bottom wells. Raw adherence (RawAd) was determined by counting PMN adhered to the bottom of the membrane in 5 microscope fields per well. Relative adherence (RelAd) was calculated as (RawAd test well) / (RawAd negative control well) × 100%. Data were analyzed using the Glimmix procedure of SAS with the fixed effects of HEPES, CO2, chemoattractant and all interactions and random effects of date and cow within date. RawAd and RelAd were both affected by CO2 (P = 0.03; 0.002, respectively) and chemoattractant (P = <.0001; 0.0002, respectively). The presence of CO2 caused a decrease in both RawAd and RelAd. For both RawAd and RelAd, chemotaxis towards C5a or IL-8 did not differ but both were greater than chemotaxis to controls. RawAd was also affected by CO2×chemoattractant (P = 0.02) and HEPES×CO2 (P = 0.0001). RawAd for C5a or IL-8 wells was approximately twice that of control wells in the absence of CO2, but was not significantly different from controls in the presence of CO2. RawAd decreased in the presence of CO2 when HEPES was not present but was unaffected by CO2 in the presence of HEPES. These results suggest that incubating PMN with CO2 reduces the chemotactic response and that HEPES partially ameliorated this effect.
neutrophils, chemotaxis, bovine