Fetal calf serum solution preparation

Prepare competitor protein buffer detergent solution 4 mg/mL normal goat globulin or other competitor protein, (such as fetal calf serum, bovine serum albumin, bovine plasma, and so forth), and 0.1% saponin (Sigma, St. Louis, MO) in phosphate-buffered saline (NGG-sap-PBS). 

The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 10 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100 /U.1) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a rosette or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted. 

EBSS/fetal calf serum (EBSS/FCS) One day prior to use, prepare a solution of EBSS with 10% FCS by adding 2 ml FCS to 8 ml EBSS. Filter and allow to equilibrate until use in a tube with a loosened lid at 37°C in a 7% CO2 incubator. 

Chang s human conjunctival cells with a population of lOVml were cultured in Eagle s minimum essential medium supplemented with 10% fetal calf serum for 48hrs in 5% CO2 in a 37 C incubator. Cell sheets were exposed to various concentrations of amphotericin B (Bristol-Myers Squibb Co., purity 100%), miconazole (Janssen Pharmaceutica Co., purity 100%), and fluconazole (Pfizer Pharmaceuticals Inc. Co., purity 100%) for 2 or 4 min. Test drug concentrations were adjusted to clinical use. Cells were prepared, immediately after exposure to the test solutions, for scanning electron microscopy and flow cytometry or cultured for a further 24 hrs to examine cell growth. 

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