Combined use of several mitogens for mitotic stimulation to human lymphocytes

Several different type of mitogens, such as phytohemagglutinin (PHA) including PHA-P, -M, -W, -C, -L and -E+L, pokeweed mitogen (PWM), Concanavalin A (Con-A), lipopolysaccharide (LPS), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were compared their mitotic stimulating effects to human lymphocytes in single use and also combined use of these mitogens. In the tests with single use of each mitogen, PHA was confirmed to be the most superior mitogen. Of several different types of PHA, PHA-C, -L and -E+L were not appropriate to stimulate human lymphocytes, though PHA-P, -M and -W induced sufficiently mitoses. The most appropriate concentration of PWM was 50 micrograms/ml, but the highest mitotic index reached at 5 days after the initiation of culture. Con-A did not induce sufficient mitoses, and LPS, WGA and SBA were inadequate to stimulate human lymphocytes in the single use. In the combined use of two mitogens, there was no reliable combination to enhance mitotic stimulation. When three different mitogens were used simultaneously, only one combination with PHA-M, PWM and SBA effectively enhanced the mitotic index when compared to the single use. In the confirmatory experiment for this combination carried out on 5 different donors, the enhanced effect to mitotic index was recognized in two out of three donors with low response to PHA, but the low level of mitotic indices was seen in remaining 2 donors with high response to PHA. This finding suggests that this combination may overcome a disadvantage in some of the low responders to PHA.