Sepracell-MN for mononuclear cell separation from cattle: effect on mitogen-and antigen-induced lymphocyte blastogenesis.
Mononuclear leukocytes (MNC) were separated from heparinized and EDTA-treated whole bovine blood by centrifugation after mixing with a commercial colloidal silica preparation (Sepracell-MN (S-MN]. Cell yields and lymphocyte blast transformation (LBT) to pokeweed mitogen (PWM), phytohaemagglutinin (PHA), concanavalin A (Con A), and Brucella abortus antigens were tested against MNC obtained from heparinized whole blood using Ficoll-Hypaque (FH). Separation with S-MN was more rapid and less labor intensive than separation with FH. There was a higher average total yield of MNC but a lower percentage of monocytes in the FH- than in the S-MN-separated MNC. In mitogen-induced LBT assays, MNC responded comparably to each mitogen regardless of the separation technique or anticoagulant used, and a cell concentration effect was demonstrated. In general, FH-separated MNC responded greater to PWM than did S-MN/EDTA separated MNC, but S-MN/heparin separated MNC had the greatest LBT responses to PWM. Overall, S-MN/EDTA separated MNC had the greatest responses to PHA, and responses to Con A were variable among experiments with respect to the separation technique. In antigen-induced LBT assays, two B. abortus antigens were used: a heat-killed strain S1119 (HKA) and a gamma-irradiated strain 19 (gamma BA). The LBT responses of three steers vaccinated with live B. abortus strain 19 were compared with three nonvaccinated steers in three separate experiments. Using HKA, FH separation resulted in an overall greater LBT response for vaccinates than nonvaccinates and a greater differential between responses of vaccinates and nonvaccinates than did S-MN derived MNC regardless of the anticoagulant used. Using gamma BA, FH produced the most responsive MNC in one experiment and S-MN/heparin produced the most responsive MNC in the other. At the highest cell concentration tested, FH-separated MNC had the greatest LBT responses for vaccinated calves, but differences between S-MN- and FH-separated MNC responses were not significantly different (P greater than 0.05). In conclusion, S-MN is a rapid and simple technique for separation of MNC from bovine blood. The technique produces an adequate cell population for mitogen-induced LBT studies; however, FH-separated MNC were generally more responsive in the B. abortus-induced LBT assay.
Confer
A W
AW
Department of Veterinary Pathology, Oklahoma State University, Stillwater.
Wyckoff
J H
JH
Oltjen
S L
SL
Oltjen
J W
JW
D000818:Animals; D000941:Antigens / Q000008:administration & dosage; D002417:Cattle / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D008934:Mitogens / Q000494:pharmacology; D012822:Silicon Dioxide
D000941:Antigens; D008934:Mitogens; C062165:Sepracell-MN; D012822:Silicon Dioxide