Standardization of bovine E-rosette assay and enumeration of lymphocyte subpopulations.

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Abstract
To standardize the E-rosette formation by bovine lymphocytes, the effects of pretreatments of sheep erythrocytes, breed and individual differences of sheep erythrocyte, and dextran concentration in rosette medium influencing E-rosettes were investigated, and also E-rosettes were examined with double markers. Different pretreatments of sheep erythrocytes, with neuraminidase (EN), 2-aminoethyl isothiouronium bromide hydrobromide (EAET) formed significantly higher percentages of rosette forming cells (RFC) (P < 0.01, < 0.1) compared with the rosette values for untreated (E) sheep erythrocytes. No significant differences in the percentages of E-rosettes were observed between 2 breeds (Suffolk (N = 9), Corriedale (n = 7)) and 1 Crossbreed (n = 5); however, individual differences were observed. Standardized method of bovine E-rosette assay (EAET-dextran) was 400 .mu.l of lymphocyte suspension (3-4 .times. 106/ml) in Eagle''s minimum essential medium containing 20% inactivated fetal calf serum (MEM-FCS); this was mixed with 400 .mu.l of a 1% sheep erythrocytes (pretreated 0.143 M AET) suspended in 8% dextran (MW 177000), incubated at 37.degree. C for 30 min and centrifuged at 100 g for 5 min, and then incubated at 4.degree. C overnight. In double marker experiments, 3% (mean) (range 0-6%) of RFC were detected which exhibited both B and T cell markers. Using the standardized E-rosette assay (EAET-dextran), Surface Ig (SIg) immunofluorescence assay and yeast ingesting method it was possible to differentiate 90.2 .+-. 6.5% (mean .+-. SD) (range 83-98%) of bovine peripheral blood mononuclear cells from 8 normal cows into T cells (61.4 .+-. 3.9%, 56-68%), B cells (22.8 .+-. 4.1%, 16-30%) and monocytes (6.1 .+-. 2.6%, 4-10%).