Thick filter neutrophil chemotaxis performed in the absence of albumin.
Migration through a filter is a technique widely used for the study of cell chemotaxis. Since the original description of the technique by Boyden in 1962 (J. Exp. Med. 115, 453) using neutrophils and thick filters prepared from cellulose nitrate, albumin has been required in the incubation medium to support chemotaxis. However, the binding to albumin of compounds affecting chemotaxis can reduce their free concentration. We developed two procedures for studying neutrophil chemotaxis under reduced or albumin-free conditions. In one, cellulose nitrate filters were pretreated with albumin by a novel procedure and chemotaxis was carried out in albumin-free medium. As tested with the chemoattractant LTB4 and human neutrophils, the procedure resulted in full chemotaxis, measured by the number of cells crossing the filter, with an EC50 of 0.43 nM for LTB4. The LTB4-receptor antagonist LY 223982 inhibited the chemotactic action of LTB4 with a Ki of 62 nM in the albumin-pretreated filter system, thus showing 58 times greater potency than in medium containing 0.5% albumin. The second procedure makes use, for the first time, of a relatively new filter (Hydrophilic Durapore). This filter has the same dimensions and pore rating of the cellulose nitrate filter but did not require pretreatment with albumin to support chemotaxis in the albumin-free medium. LTB4 stimulated neutrophil chemotaxis across this filter with an EC50 of 0.29 nM. LY 223982 had a Ki of 11 nM, thus exhibiting a potency even greater than in the albumin-pretreated cellulose nitrate filter system. fMLP and C5a also stimulated chemotaxis in the absence of albumin. These results suggest that albumin is not obligatory for neutrophil chemotaxis through thick filters. The role of albumin in the chemotaxis assay using cellulose nitrate filters may be to counteract the adherence of cells or chemotactic agents to the filters.
Psychoyos
S
S
Research Department, Pharmaceuticals Division, Ciba-Geigy Corporation, Summit, NJ 07901.
Uziel-Fusi
S
S
Morrissey
M M
MM
Ganu
V
V
Smith
C W
CW
D000418:Albumins / Q000494:pharmacology*; D001577:Benzophenones / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003101:Collodion; D015936:Complement C5a / Q000494:pharmacology; D005374:Filtration; D006801:Humans; D007975:Leukotriene B4 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology*
D000418:Albumins; D001577:Benzophenones; C065916:LY 223982; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D003101:Collodion